METHODS AND KITS FOR DETERMINING CELL SECRETED BIOMOLECULES
20220145355 · 2022-05-12
Inventors
Cpc classification
C12M41/00
CHEMISTRY; METALLURGY
C12Q2537/143
CHEMISTRY; METALLURGY
C12Q2537/143
CHEMISTRY; METALLURGY
C12Q1/6876
CHEMISTRY; METALLURGY
International classification
C12M1/12
CHEMISTRY; METALLURGY
C12M1/34
CHEMISTRY; METALLURGY
C12Q1/6876
CHEMISTRY; METALLURGY
Abstract
The invention inter alia pertains to methods and kits for analysis of one or more cell released biomolecules. Furthermore, the invention relates to a plurality of sequenceable products comprising different sequence elements. The described technology is useful for a variety of applications, in particular biomolecule analysis applications, e.g. for obtaining biomolecule release profiles of single cells in a multiplexed manner, wherein the cells are provided in a matrix
Claims
1. A method for analyzing one or more cell released biomolecules, comprising providing a cell-laden matrix, wherein the cell-laden matrix comprises at least one cell that releases one or more biomolecules of interest, wherein the method comprises the following steps: a) providing a capture matrix, wherein the capture matrix comprises one or more types of capture molecules, wherein each type of capture molecule binds a biomolecule of interest; b) incubating the cell-laden matrix to allow release of the one or more biomolecules of interest and binding the one or more biomolecules of interest to the one or more types of capture molecules of the capture matrix; c) adding one or more types of detection molecules, wherein each type of detection molecule specifically binds a biomolecule of interest, and wherein each type of detection molecule comprises a barcode label which comprises a barcode sequence (B.sub.S) indicating the specificity of the detection molecule; d) generating a sequenceable reaction product which comprises at least (i) the barcode sequence (B.sub.S), and (ii) a barcode sequence (B.sub.T) for indicating a time information, and/or (iii) a barcode sequence (B.sub.P) for indicating a position information, and (iv) optionally a unique molecular identifier (UMI) sequence, wherein generation of the sequenceable reaction product comprises the use of at least one oligonucleotide, optionally a primer, that is capable of hybridizing to the barcode label of the at least one type of detection molecule or the use of at least one oligonucleotide that is ligated to the barcode label of the at least one type of detection molecule.
2. The method according to claim 1, wherein the sequenceable reaction product comprises a barcode sequence (B.sub.T) for indicating a time information and wherein n cycles of steps a) to c) and optionally step d) are performed at different time points t.sub.x, wherein n is at least 2 and x indicates the different time points, and wherein for each cycle a sequenceable reaction product is generated that differs in its barcode sequence B.sub.T from the barcode sequence B.sub.T of all other performed cycles.
3. The method according to claim 1 or 2, wherein a plurality of cell-laden matrices and capture matrices are provided in a cell culture device comprising a plurality of compartments, wherein at least one cell-laden matrix and at least one capture matrix are provided within a compartment of the cell culture device.
4. The method according to claim 3, wherein the method comprises obtaining capture matrices from a plurality of compartments and transfer of the capture matrices to a device comprising a plurality of compartments.
5. The method according to claim 3 or 4, wherein at least one cycle of steps a) to d) is performed for a plurality of cell-laden matrices comprised in different compartments and wherein the sequenceable reaction product that is generated in step d) comprises a barcode sequence B.sub.P for indicating position information of a cell-laden matrix analysed, wherein a sequenceable reaction product is generated for a cell-laden matrix comprised in a compartment that differs in its barcode sequence B.sub.P from the barcode sequence B.sub.P of the sequenceable reaction product(s) generated for a cell-laden matrix comprised in another compartment.
6. The method according to claim 5, wherein the barcode sequence B.sub.P is introduced into the sequenceable reaction product via an oligonucleotide that is used in step d), wherein the oligonucleotide comprising the barcode sequence B.sub.P is a primer that is used in an amplification reaction.
7. The method according to one or more of claims 1 to 6, comprising analyzing y different biomolecules of interest using different types of capture molecules and different types of detection molecules, wherein y is at least 2 and wherein the barcode label of each type of detection molecule that binds a biomolecule of interest differs in its barcode sequence B.sub.S from the barcode sequence B.sub.S of all other types of detection molecules that bind a different biomolecule of interest.
8. The method according to one or more of claims 1 to 7, wherein step d) comprises performing an amplification reaction using a primer or primer combination, optionally wherein step d) additionally comprises extending the barcode label using an adaptor barcode oligonucleotide capable of hybridizing to the barcode label as template, whereby an extended barcode label is provided in advance of the amplification reaction.
9. The method according to one or more of claims 1 to 8, wherein the method comprises e) sequencing the generated sequenceable reaction product(s), optionally wherein the method comprises pooling sequenceable reaction products generated in step d) from different cycles and/or generated from different compartments and sequencing the obtained pool.
10. The method according to one or more of claims 1 to 9, wherein step d) comprises (aa) hybridizing at least one oligonucleotide to the barcode label of at least one type of detection molecule and extending said barcode label using the hybridized oligonucleotide as template thereby obtaining an extended barcode label attached to the detection molecule that additionally comprises sequence information of the hybridized oligonucleotide that was used as template, optionally wherein step d) further comprises (bb) performing an amplification reaction with a primer or primer combination using the extended barcode label and/or the reverse complement thereof as template, wherein preferably, the extended barcode label is used as template.
11. The method according to one or more of claims 1 to 10, wherein generation of the sequenceable reaction product in step d) comprises the use of (i) at least one oligonucleotide, optionally a primer, and/or (ii) a primer combination, wherein the at least one oligonucleotide and/or the primer combination includes one or more sequence elements selected from the group consisting of a barcode sequence (B.sub.T) for indicating a time information, a barcode sequence (B.sub.P) for indicating position information of a cell-laden matrix, a unique molecular identifier (UMI) sequence, optionally wherein the UMI sequence has a length of up to 40 nucleotides, preferably 4-20 nucleotides, and an adapter sequence (AS) for sequencing, wherein the one or more sequence elements B.sub.T, B.sub.P, UMI and/or AS, if included, are located 5′ of the sequence region of the oligonucleotide and/or primer that is capable of hybridizing to the barcode label of the detection molecule or the reverse complement thereof.
12. The method according to one or more of claims 1 to 11, wherein the barcode label attached to a detection molecule and/or the extended barcode label obtained according the method of claim 9 to 11 comprises (i) the barcode sequence (B.sub.S) indicating the specificity of the detection molecule; (ii) one or more primer target sequences; (iii) optionally a barcode sequence (B.sub.T) indicating a time information; (iv) optionally a unique molecular identifier (UMI) sequence; and (v) optionally an adapter sequence (1).
13. The method according to one or more of claims 1 to 12, wherein step d) comprises per Variant A (aa) adding an adaptor barcode oligonucleotide capable of hybridizing to the barcode label of at least one type of detection molecule, wherein the adaptor barcode oligonucleotide comprises 5′ to the region that is capable of hybridizing to the barcode label a unique molecular identifier (UMI) sequence, and extending the barcode label using the hybridized adaptor barcode oligonucleotide as template thereby obtaining an extended barcode label; wherein preferably step d) further comprises (bb) performing an amplification reaction with a primer or primer combination using the extended barcode label and/or the reverse complement thereof as template; or per Variant B (aa) adding an adaptor barcode oligonucleotide capable of hybridizing to the barcode label of at least one type of detection molecule, wherein the adaptor barcode oligonucleotide comprises 5′ to the region that is capable of hybridizing to the barcode label (i) a barcode sequence (B.sub.P) for indicating a position information and (ii) preferably a unique molecular identifier (UMI) sequence, and extending the barcode label using the hybridized adaptor barcode oligonucleotide as template thereby obtaining an extended barcode label; wherein preferably, step d) further comprises (bb) performing an amplification reaction with a primer or primer combination using the extended barcode label and/or the reverse complement thereof as template; or per Variant C (aa) adding an adaptor barcode oligonucleotide capable of hybridizing to the barcode label of at least one type of detection molecule, wherein the adaptor barcode oligonucleotide comprises 5′ to the region that is capable of hybridizing to the barcode label (i) a barcode sequence (B.sub.T) for indicating a time information and/or (ii) a unique molecular identifier (UMI) sequence, and extending the barcode label using the hybridized adaptor barcode oligonucleotide as template thereby obtaining an extended barcode label; wherein preferably step d) further comprises (bb) performing an amplification reaction with a primer or primer combination using the extended barcode label and/or the reverse complement thereof as template.
14. The method according to any one of claims 1 to 13, wherein step d) comprises (aa) adding an adaptor barcode oligonucleotide, wherein the adaptor barcode oligonucleotide comprises an adaptor sequence (1).sub.R that is reverse complementary to an adapter sequence (1) of the barcode label of the detection molecule, wherein the adaptor barcode oligonucleotide additionally comprises at least one, at least two, at least three or all sequence elements selected from the group consisting of a barcode sequence (B.sub.T) for indicating a time information, a barcode sequence (B.sub.P) for indicating a position information, a unique molecular identifier (UMI) sequence, and a primer target sequence, wherein these one or more sequence elements are located 5′ of the adaptor sequence (1).sub.R and extending the barcode label using the hybridized adaptor barcode oligonucleotide as template thereby obtaining an extended barcode label.
15. The method according to claim 9 to 14, wherein step d) comprises performing an amplification reaction with a primer or primer combination comprising a barcode sequence (B.sub.P) for indicating position information, optionally an adapter sequence (AS) for sequencing, optionally a barcode sequence (B.sub.T) for indicating a time information, wherein the one or more sequence elements B.sub.P, AS, and/or B.sub.T if included in the primer or a primer of the primer combination, are located 5′ of the sequence region of the primer that is capable of hybridizing to the optionally extended barcode label or the reverse complement thereof.
16. The method according to claim 15, wherein the templates comprised in different compartments of a device are contacted with a different subtype of the primer or primer combination, wherein the different subtypes of the primer or primer combination differ in their barcode sequence B.sub.P that indicates the position information of an individual compartment, wherein preferably, the subtypes of the primer or primer combination are identical except for the barcode sequence B.sub.P that is unique for each subtype.
17. The method according to claim 16, wherein the amplification in step d) is performed by contacting the templates comprised in different compartments of a device with different primer combinations, wherein one primer of the primer combination is the same for all templates comprised in different compartments of the device and the other primer of the primer combination differs in the barcode sequence B.sub.P that indicates the position information of an individual compartment.
18. The method according to one or more of claims 1 to 17, wherein the barcode sequence B.sub.T is provided in the barcode label or the extended barcode label and wherein step d) comprises pooling barcode labels or extended barcode labels provided at different time points and comprising different barcode sequences B.sub.T in a compartment prior to performing an amplification reaction.
19. The method according to one or more of claims 1 to 18, having one or more of the following features a. the matrix comprising at least one cell has one or more of the following characteristics: (i) the matrix material is provided by a hydrogel; (ii) the matrix is three-dimensional; (iii) the matrix is a particle, optionally a hemi-spherical particle or preferably a spherical particle; (iv) the matrix has a diameter of ≤1000 μm, such as ≤800 μm, ≤600 μm, or ≤400 μm, preferably ≤200 μm, such as 5 μm to 150 μm; and/or (vi) the matrix has a volume of ≤200 μl, such as ≤100 μl, ≤50 μl, ≤10 μl, ≤1 μl, ≤0.5 μl, ≤300 nl, <200 nl, ≤100 nl, <50 nl or ≤5 nl, preferably 0.05 pl to 2000 pl; b. the capture matrix comprising the one or more types of capture molecules has one or more of the following characteristics: (i) it is a polymer matrix, optionally comprising or consisting of polyacrylamide (PMA), polyactic acid (PLA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), polyoxazoline (POx), and polystyrene (PS). (ii) the matrix material is provided by a hydrogel; (iii) the matrix is three-dimensional; (iv) the matrix is a particle, preferably a spherical particle; and/or (v) the matrix has a diameter of ≤1000 μm, such as ≤800 μm, ≤600 μm or ≤400 μm, preferably ≤200 μm, such as 5 μm to 150 μm; and/or c. the cell-laden matrix and the capture matrix are provided in proximity within a compartment of a device or the cell-laden matrix and the capture matrix are provided in separate compartments, wherein the separate compartments are in fluid communication with each other or can be brought in fluid communication with each other so that the released biomolecules of interest can contact the capture matrix.
20. The method according to one or more of claims 1 to 19, wherein the matrix of the cell-laden matrix is a hydrogel which has one or more of the following characteristics: a. the hydrogel comprises cross-linked hydrogel precursor molecules of the same type or of different types; b. the hydrogel is composed of at least two different polymers with different structures as hydrogel precursor molecules, wherein optionally, at least one polymer is a copolymer; c. the hydrogel is formed using at least one polymer which has a linear structure and at least one polymer which has a multiarm or star-shaped structure; d. the hydrogel is formed using a t least one polymer of formula (P1) ##STR00004## wherein R is independently selected from a hydrogen atom, a hydrocarbon with 1-18 carbonatoms (preferably CH.sub.3, —C.sub.2H.sub.5,), a C.sub.1-C.sub.25-hydrocarbon with at least one hydroxy group, a C.sub.1-C.sub.25-hydrocarbon with at least one carboxy group, (C.sub.2-C.sub.6)alkylthiol, (C.sub.2-C.sub.6)alkylamine, protected (C.sub.2-C.sub.6)alkylamine (preferably-(CH.sub.2).sub.2-6—NH—CO—R (with R=tert-Butyl, perfluoroalkyl)), (C.sub.2-C.sub.6)alkylazide, polyethylene glycol, polylactic acid, polyglycolic acid, polyoxazoline, or wherein R is a residue R.sup.4 Y is a moiety containing at least one graft, comprising at least one residue R.sup.4, T.sub.1 is a terminating moiety, which may contain a residue R.sup.4, T.sub.2 is a terminating moiety, which contains a residue R.sup.4, p is an integer from 1 to 10, n is an integer greater than 1 and preferably, below 500, m is zero or an integer of at least, preferably greater than 1, and preferably, below 500, the sum n+m is greater than 10, x is independently 1, 2 or 3, preferably x is independently 1 or 2, most preferably x is 1, R.sup.4 independently comprise at least one functional group for crosslinking and/or for binding biologically active compounds, and optionally comprising a (preferably degradable) spacer moiety connecting said functional group with the binding site to the respective moiety of the structure of formula (P1), wherein the entirety of all m-fold and n-fold repeating units are distributed in any order within the polymer chain and wherein optionally, the polymer is a random copolymer or a block copolymer.
21. The method according to one or more of claims 1 to 20, wherein the method is performed by utilizing a cell culture device, which preferably is a microfabricated cell culture device, wherein the device has one or more of the following features: i) at least one compartment for accommodating at least one, preferably at least two matrices, including at least one capture matrix and/or at least one cell-laden matrix; ii) at least one compartment that is capable of being switched between an isolated and an open state, wherein the isolated state corresponds to a state at which fluid that is present in the compartment is in no contact with fluid not present in the compartment and wherein the open state corresponds to a state at which fluid that is present in the compartment is in contact with fluid not present in the compartment; iii) a compartment for accommodating at least one matrix, preferably two matrices, wherein a microfabricated geometry for matrix immobilization is present suitable for positioning the at least one matrix; iv) a plurality of compartments for accommodating at least one matrix, preferably provided by an array of compartments; v) a microfabricated valve capable of switching the compartment to an open or closed state; vi) a microfabricated valve, comprising a first channel, a second channel, a connection channel connecting the first channel and the second channel, a valve portion arranged within the connection channel, wherein the valve portion is adapted to selectively open and close the connection channel; vii) a microfabricated valve comprising at least three layers, wherein a first channel is located within a first layer; a second channel is located within a third layer; a valve portion is located within a second layer; the second layer is arranged between the first and the third layer; viii) a microfabricated valve wherein a first channel comprises a microfabricated geometry for matrix immobilization suitable for positioning at least one matrix being contained in a fluid which flows through the first channel, wherein the microfabricated geometry for matrix immobilization is arranged within the first channel in such a way that a fluid flow can be reduced by the microfabricated geometry for matrix immobilization, in particular, the microfabricated geometry for matrix immobilization narrows the cross section of the channel; and/or wherein a second channel comprises a microfabricated geometry for matrix immobilization suitable for positioning particles being contained in a fluid which flows through the second channel, wherein the microfabricated geometry for matrix immobilization is arranged within the second channel in such a way that a fluid flow can be reduced by the microfabricated geometry for matrix immobilization, in particular, the microfabricated geometry for matrix immobilization narrows the cross section of the channel; and/or ix) a fluid reservoir and fluid channels for providing fluid to the compartment.
22. The method according to one or more of claims 1 to 21, wherein the method is performed by utilizing a cell culture device, which preferably is a microfabricated cell culture device, wherein the device comprises one or more of the following features: i) at least one matrix is releasably positioned by a preferably microfabricated geometry for matrix immobilization inside a compartment; ii) at least one matrix is releasably positioned by a preferably microfabricated geometry for matrix immobilization inside a compartment, wherein the geometry for matrix immobilization has one or more of the following characteristics: it is capable of positioning the cell-laden matrix and the capture matrix in proximity; it is capable of positioning at least two cell-laden matrix and the capture matrix in proximity; iii) at least one cell-laden matrix and at least one capture matrix are positioned by a preferably microfabricated geometry for matrix immobilization inside a compartment, wherein the compartment accommodating the at least one cell-laden matrix is different from the compartment accommodating the at least one capture matrix and wherein both compartments can be switched to be either in fluid contact with other or to be in no fluid contact with each other; and/or iv) it comprises a trapping geometry comprising a valve arrangement adapted to provide a fluid passing through a microfabricated geometry for matrix immobilization wherein the valve arrangement is adapted to selectively change the direction of fluid passing the microfabricated geometry for matrix immobilization, in particular wherein a fluid a first direction urging the at least one matrix into the microfabricated geometry for matrix immobilization and a fluid in the second direction urging the at least one matrix out of the microfabricated geometry for matrix immobilization, and in particular fluid in the second direction delivering the at least one matrix in direction of an exit section.
23. The method according to one or more of claims 1 to 22, wherein the provided cell-laden matrix and capture matrix are provided with a fluid, preferably a fluid that is immiscible with water, wherein said matrices, provided with said fluid, are preferably generated by utilizing a cell culture device, which preferably is a microfabricated cell culture device, and preferably by (i) releasably positioning the cell-laden matrix and the capture matrix by a preferably microfabricated geometry for matrix immobilization inside a compartment, wherein the compartment comprises a first fluid, preferably an aqueous fluid; (ii) removing the first fluid from the compartment and replacing the first fluid by a second fluid that provides said fluid, wherein said fluid is preferably immiscible with water; and (iii) optionally, removing the second fluid from the compartment and replacing it by the first fluid or a third fluid, that is preferably immiscible with the second fluid.
24. The method according to one or more of claims 1 to 23, wherein the cell-laden matrix is incubated to allow release of one or more biomolecules of interest before providing the capture matrix in step a), wherein after providing the capture matrix, one or more biomolecules of interest are specifically bound by the one or more types of capture molecules of the capture matrix; wherein preferably, the cell-laden matrix is provided in a defined volume of a fluid, preferably a fluid that is immiscible with water, and wherein the capture matrix is provided in a defined volume of the same type of fluid, and wherein after contacting the cell-laden matrix and the capture matrix said fluids of the same type merge to provide a defined volume of fluid that is shared by the cell-laden matrix and the capture matrix.
25. A kit comprising a) one or more types of detection molecules, wherein each type of detection molecule specifically binds a biomolecule of interest, and wherein each type of detection molecule comprises a barcode label which comprises a barcode sequence (B.sub.S) indicating the specificity of the detection molecule; and b) at least one oligonucleotide, optionally a primer, that is preferably capable of hybridizing to the barcode label of the at least one type of detection molecule.
26. The kit according to claim 25, wherein the oligonucleotide comprises at least one sequence element selected from the group consisting of (i) a barcode sequence (B.sub.T) for indicating a time information, (ii) a barcode sequence (B.sub.P) for indicating a position information, and (iii) a unique molecular identifier (UMI) sequence.
27. The kit according to claim 25 or 26, wherein the kit has one or more of the following characteristics: a. it comprises an adaptor barcode oligonucleotide capable of hybridizing to the barcode label of at least one type of detection molecule, wherein the adaptor barcode oligonucleotide comprises 5′ to the region that is capable of hybridizing to the barcode label (i) a barcode sequence (B.sub.T) for indicating a time information, a barcode sequence (B.sub.P) for indicating a position information, and/or (ii) a unique molecular identifier (UMI) sequence; b. it comprises an adaptor barcode oligonucleotide, wherein the adaptor barcode oligonucleotide comprises an adaptor sequence (1).sub.R that is reverse complementary to an adapter sequence (1) of the barcode label of the detection molecule, wherein the adaptor barcode oligonucleotide additionally comprises at least one, at least two, at least three or all sequence elements selected from the group consisting of a barcode sequence (B.sub.T) for indicating a time information, a barcode sequence (B.sub.P) for indicating a position information, a unique molecular identifier (UMI) sequence, and a primer target sequence, wherein these one or more sequence elements are located 5′ of the adaptor sequence (1).sub.R; c. a primer or primer combination comprising one or more of the following a barcode sequence (B.sub.P) for indicating position information, a barcode sequence (B.sub.T) for indicating a time information, an adapter sequence (AS) for sequencing, wherein the one or more sequence elements B.sub.P, AS, and/or B.sub.T if included in the primer or a primer of the primer combination, are located 5′ of the sequence region of the primer that is capable of hybridizing to the optionally extended barcode label or the reverse complement thereof; and/or d. the barcode label of the one or more types of detection molecules comprises (i) the barcode sequence (B.sub.S) indicating the specificity of the detection molecule; (ii) one or more primer target sequences; (iii) optionally a barcode sequence (B.sub.T) indicating a time information; (iv) optionally a unique molecular identifier (UMI) sequence; and (v) optionally an adapter sequence (1).
28. The kit according to any one of claims 25 to 27, wherein the kit comprises at least one set of oligonucleotides selected from the following group: a) set 1 comprising: a. a barcode label attached to the detection molecule comprising: i. optionally a cleavable linker/spacer, ii. optionally a first primer binding sequence (1), iii. a barcode sequence B.sub.S, iv. an adaptor sequence (1); b. an adaptor barcode oligonucleotide comprising: i. an adaptor sequence (1).sub.R, ii. a unique molecular identifier (UMI) sequence, iii. a second primer binding sequence (2).sub.R, c. a forward primer comprising: i. a primer sequence (1), ii. a barcode sequence B.sub.P, iii. an adaptor sequence for sequencing (AS); d. a reverse primer comprising: i. a primer sequence (2).sub.R, ii. a barcode sequence B.sub.T, iii. an adaptor sequence for sequencing (AS); b) set 2 comprising: a. a barcode label attached to the detection molecule comprising: i. optionally a cleavable linker/spacer, ii. a first primer binding sequence (1), iii. a barcode sequence B.sub.S, iv. an adaptor sequence (1); b. an adaptor barcode oligonucleotide comprising: i. an adaptor sequence (1).sub.R, ii. a barcode sequence B.sub.P, iii. a unique molecular identifier (UMI) sequence, iv. a second primer binding sequence (2).sub.R; c. a forward primer comprising: i. a primer sequence (1), ii. a barcode sequence B.sub.T, iii. an adaptor sequence for sequencing (AS); d. a reverse primer comprising: i. a primer sequence (2).sub.R, ii. an adaptor sequence for sequencing (AS); c) set 3 comprising: a. a barcode label attached to the detection molecule comprising: i. optionally a cleavable linker/spacer, ii. a first primer binding sequence (1), iii. a barcode sequence B.sub.S, iv. an adaptor sequence (1); b. an adaptor barcode oligonucleotide comprising: i. an adaptor sequence (1).sub.R, ii. a barcode sequence B.sub.T, iii. a unique molecular identifier (UMI) sequence, iv. a second primer binding sequence (2).sub.R; c. a forward primer comprising: i. a primer sequence (1), ii. a barcode sequence B.sub.P, iii. an adaptor sequence for sequencing (AS); d. a reverse primer comprising: i. a primer sequence (2).sub.R, ii. an adaptor sequence for sequencing (AS); d) set 4 comprises: a. a barcode label attached to the detection molecule comprising: i. optionally a cleavable linker/spacer, ii. a first primer binding sequence (1), iii. a barcode sequence B.sub.S, iv. a unique molecular identifier (UMI) sequence, v. a barcode sequence B.sub.T, vi. a second primer binding sequence (2); b. a forward primer comprising: i. a primer sequence (1), ii. a barcode sequence B.sub.P, iii. an adaptor sequence for sequencing (AS); c. a reverse primer comprising: i. a primer sequence (2).sub.R, ii. an adaptor sequence for sequencing (AS).
29. The kit according to any one of claims 25 to 28, wherein the kit comprises at least one of the following a. one or more types of capture molecules, wherein each type of capture molecule binds a biomolecule of interest, wherein preferably, the one or more types of capture molecules provided in the kit bind the same biomolecules of interest as the one or more types of detection molecules comprised in the kit; b. one or more polymers for providing the matrix for the cells and/or the capture matrix, wherein preferably the polymer is capable of forming a hydrogel; c. a composition, preferably a solution, containing capture matrices; d. polymerase and/or dNTPs; and/or e. a wash solution.
30. The kit according to any one of claims 25 to 29, wherein the kit comprises a device with a plurality of compartments, preferably a multi-well plate, wherein said device has one or more of the following characteristics: a. compartments of the device comprise an oligonucleotide, preferably an adaptor barcode oligonucleotide and/or a primer or primer combination, as defined in claims 25 to 29; b. compartments of the device comprise at least one set as defined in claim 28; c. compartments comprising an oligonucleotide, preferably an adaptor barcode oligonucleotide and/or a primer or primer combination, as defined in any one of claims 25 to 29, furthermore comprise reagents for performing an extension and/or amplification reaction; and/or d. the device is selected from a 96, 384 or 1536 well plate.
31. A plurality of sequenceable products, wherein each sequenceable product comprises at least the following sequence elements (i) a barcode sequence (B.sub.S) for indicating a specificity, and (ii) a barcode sequence (B.sub.T) for indicating a time information, and/or (iii) a barcode sequence (B.sub.P) for indicating a position information, and (iv) optionally a unique molecular identifier (UMI) sequence.
32. The plurality sequenceable products according to claim 31, wherein the sequenceable products differ from each other in one or more of the comprised sequence elements (i) to (iv).
33. The plurality of sequenceable products as defined in claim 31 or 32, having one or more of the following features: a. the number of sequenceable products comprising different sequence elements B.sub.S, B.sub.T and/or B.sub.P is at least 50, preferably at least 100; b. the plurality of sequenceable products comprise at least 2 different barcode sequences B.sub.S, optionally wherein the number of different barcode sequences B.sub.S may lie in a range of 2 to 100, 5 to 50, 5 to 25, 5 to 20 or 7 to 15; c. the plurality of sequenceable products comprise at least 2 different barcode sequences B.sub.T, optionally wherein the number of different barcode sequences B.sub.T may lie in a range of 2 to 200, 5 to 50, 5 to 25, 5 to 20 or 7 to 15; and d. the plurality of sequenceable products comprise at least 2 different barcode sequences B.sub.P, optionally wherein the number of different barcode sequences B.sub.P may lie in a range of 2 to 1000, 5 to 1000, 10 to 500, 20 to 250 or 50 to 200; and/or e. wherein the UMI sequence has a length of up to 40 nucleotides, preferably 4 to 20 nucleotides.
34. The method according to one or more of claims 1-20, wherein the cell-laden matrix is provided in a compartment of a cell culture plate such that liquid that covers the cell-laden matrix can be removed or exchanged without affecting the cell-laden matrix, and wherein the cell-laden matrix comprises more than one cell and is provided by a three-dimensional hydrogel matrix, optionally having at least partially an ellipsoidal shape, preferably a plug or semi-sphere shape.
35. The method according to claim 34, wherein one or more capture matrices are provided in step a), the method having one or both of the following characteristics: incubating the cell-laden matrix to allow release of one or more biomolecules of interest before adding the provided capture matrix/matrices to the compartment of the cell culture plate to bind the one or more released biomolecules of interest to the one or more types of capture molecules of the capture matrix, wherein optionally incubating is performed for an incubation period selected from 1 h to 72 h; and/or the capture matrix is transferred to another compartment after binding the one or more biomolecules of interest to the one or more types of capture molecules of the capture matrix in step b).
36. The method according to one or more of claims 1-20 or 34-35, wherein biomolecules are analyzed time-dependently, wherein the time interval between analyses is selected from ≥10 min, ≥20 min, ≥30 min, ≥1 h, ≥2 h, ≥3 h, ≥4 h, 5 h or more, up to days 1 d, 2 d or several days, preferably selected from the range of 30-120 min.
Description
DETAILED DESCRIPTION OF EMBODIMENTS ILLUSTRATED IN THE FIGURES
[0613]
[0614]
[0615] As illustrated in A, a cell-laden matrix (1), which preferably is a hydrogel bead, and a capture matrix (2), which preferably is a hydrogel bead, are positioned in close proximity within an isolated compartment at a position X|Y of a device, which according to a preferred embodiment is a microfabricated cell culture device. The cell-laden matrix comprises in the illustrated embodiment a single cell (3), which secretes two biomolecules of interest (4a and 4b). The capture matrix (2) comprises in the illustrated embodiment two different types of capture molecules (5a and 5b), which specifically bind the biomolecules of interest (4a and 4b). The different types of capture molecules are in the illustrated embodiment provided by antibodies with different specificities against the secreted biomolecules of interest. The capture matrix preferably comprises a plurality of capture molecules of the same type to ensure efficient capture of a biomolecule of interest. The capture molecules may be provided in excess of the expected number of secreted biomolecule of interest.
[0616] In B, the cell-laden matrix (1) is incubated to allow sufficient secretion of the biomolecules of interest which diffuse from the cell-laden matrix (1) to the capture matrix (2), where a biomolecule of interest is bound by the matching type of capture molecule (see interaction pairs 4a/5a and 4b/5b). Unbound molecules may be washed away.
[0617] In C, one or more types of detection molecules are added, here two types of detection molecules (6a and 6b), wherein each type of detection molecule specifically binds a biomolecule of interest. Importantly, each type of detection molecule comprises a barcode label (7) which comprises a barcode sequence (B.sub.S) indicating the specificity of the detection molecule. Thus, the specificity of the capture molecule can be determined based on the barcode label. The barcode label may be provided by an oligonucleotide sequence that may be attached via a linker to the detection molecule. In an embodiment, the linker is provided by a photocleavable spacer. In the illustrated embodiment the different types of detection molecules are provided by antibodies which bind the biomolecule of interest at a different epitope than the antibodies used for capturing. Thereby, a complex is formed, comprising the capture molecule, the biomolecule of interest and the detection molecule (see complex 4a/5a/6a and 4b/5b/6b).
[0618] In D, a sequenceable reaction product is generated which comprises at least (i) the barcode sequence (B.sub.S), and (ii) a barcode sequence (B.sub.T) for indicating a time information, and/or (iii) a barcode sequence (B.sub.P) for indicating position information of the cell-laden matrix, and (iv) optionally a unique molecular identifier (UMI) sequence. The generation of the sequenceable reaction product comprises the use of at least one oligonucleotide, which in one embodiment is a primer, that is capable of hybridizing to the barcode label of the at least one type of detection molecule. As is described herein and also illustrated in the subsequent figures, step D may comprise several substeps, including transfer steps.
[0619] One embodiment of step D that is schematically illustrated in
[0620] The capture matrix with the detection molecules, that comprise the barcode labels, which were optionally extended as described above in step D (aa), may be obtained from the compartment and can be transferred to a pre-defined compartment, such as a pre-defined well, of a different device. The transfer may occur using the RFCP-mechanism that is described elsewhere herein. The capture matrix with the (optionally extended) barcode labels may be e.g. transferred into a well of another format such as a 96-well plate. In embodiments, the transfer of the capture matrix occurs prior to step D, e.g. after capturing the biomolecules of interest in step B and/or after binding the detection molecules in step C. The removal of the capture matrix which comprises the complexes comprising the capture molecule, the biomolecule of interest and the detection molecule from the compartment leaves the cell-laden matrix in the compartment. As is illustrated in F, a “fresh” capture matrix may be added/loaded into the compartment and a new cycle may be performed at a different time-point. The steps may be repeated at several time-points.
[0621] Preferably, D comprises performing an amplification reaction using a primer or primer combination. In the illustrated embodiment, such amplification reaction is performed after performing step D (aa). The amplification reaction is indicated in
[0622] The amplification products may then be sequenced in step E. As is described herein, the method according to the present invention provides multiple pooling options, allowing to make the sequencing very cost and time efficient.
[0623]
[0624]
[0625]
[0626]
[0627] A: Illustrates a schematic scaffold structure of the core elements. The sequenceable reaction product comprises:
[0628] (i) the barcode sequence (B.sub.S) for indicating the specificity of the detection molecule (specificity information); and
[0629] (ii) a barcode sequence (B.sub.T) for indicating a time information (e.g. time-point) in which certain biomolecules of interest have been secreted/detected (time information); and/or
[0630] (iii) a barcode sequence (B.sub.P) for indicating a position information; and
[0631] (iv) optionally a unique molecular identifier (UMI) sequence, for quantifying the number of detection molecules that have bound a biomolecule of interest (information about quantity); and
[0632] (v) optionally an adapter sequence (AS) for sequencing.
[0633] As is also apparent from the illustrated embodiments, the order of the barcode sequences in the sequenceable reaction product may vary. Furthermore, additional sequence stretches (illustrated by white boxes) may or may not be present between the different barcode sequences/sequence elements.
[0634] B: The shown sequenceable reaction product can be obtained by the method depicted in
[0635]
[0636]
[0640] The illustrated order of these sequence elements is not limiting and may accordingly differ (e.g. B.sub.T, B.sub.S, UMI or UMI, B.sub.S, B.sub.T etc.). The barcode label may be attached via a linker such as a photocleavable spacer. The sequence elements B.sub.S, B.sub.T and UMI are in the illustrated embodiment flanked by primer sequences (1) and (2) which provide target sequences for the amplification primers. The barcode label may be attached to the detection molecule prior to contacting the detection molecule with the capture matrix (“off-chip”). The detection molecule binds the captured biomolecule of interest to which it specifically binds as has been explained in conjunction with
[0641]
[0644] Furthermore, it comprises primer sequences (1) and (2). The barcode label may again be attached to the detection molecule prior to contacting the capture matrix with the detection molecule. As explained above, the detection molecules bind the biomolecules of interest captured in the capture matrix. The capture matrix comprising the captured biomolecules of interest and the bound detection molecules comprising the barcode label indicating information about the specificity of the detection molecules (barcode B.sub.S), as well as indicating a quantity information (here in form of an UMI sequence) may be in one embodiment transferred to a compartment (e.g. well) of a different device, also referred to herein as collection position (e.g. collection well), for performing an amplification reaction. Subsequently, an amplification may be performed using a primer or primer combination comprising [0645] a barcode sequence B.sub.P for indicating position information, and [0646] a barcode sequence B.sub.T for indicating time information.
[0647] A primer combination in form of a primer pair may be used for amplification, wherein the forward primer comprises the barcode sequence B.sub.P and the reverse primer comprises the barcode sequence B.sub.T, or vice versa. Accordingly, the barcode sequences for indicating a time information (B.sub.T) and a position information (B.sub.P) can be added within a collection position (e.g. well), i.e. after separating the capture matrix from the cell-laden matrix. The used primers may furthermore comprise adapter sequences AS at their 5′ ends as shown in
[0648] Incorporating the quantity information (UMI sequence) as part of the barcoded label that is associated with a detection molecule (as is illustrated in
[0649]
[0650] (aa) hybridizing at least one oligonucleotide to the barcode label of at least one type of detection molecule and extending said barcode label using the hybridized oligonucleotide as template thereby obtaining an extended barcode label attached to the detection molecule that additionally comprises sequence information of the hybridized oligonucleotide that was used as template, and
[0651] (bb) performing an amplification reaction with a primer or primer combination using the extended barcode label and/or the reverse complement thereof as template.
[0652] The at least one oligonucleotide that is capable of hybridizing to the barcode label of the at least one type of detection molecule to which claim 1 refers may correspond in these embodiments to the oligonucleotide (also referred to as adaptor barcode oligonucleotide) that is capable of hybridizing to the barcode label.
[0653]
[0658] As explained above, the order of the barcode sequence B'S and the UMI sequence may vary. However, the adaptor sequence (1) is provided 3′ to these sequence elements. The barcode label, which preferably is provided by an oligonucleotide sequence that can be attached to the detection molecule via a photocleavable linker, can be attached to the detection molecule prior to contacting the capture matrix with the detection molecules. In the shown embodiment, step d) comprises a first substep (aa), wherein an adaptor barcode oligonucleotide is added, which is capable of hybridizing to the barcode label of the detection molecule. In the illustrated embodiment, the adaptor barcode oligonucleotide comprises an adaptor sequence (1).sub.R that is reverse complementary to an adapter sequence (1) of the barcode label of the detection molecule whereby it hybridizes to the barcode label. The adaptor barcode oligonucleotide may additionally comprise, as is illustrated in
[0659] In this case, only a short double-stranded region is provided upon hybridization and extension of the barcode label, which comprises the adaptor barcode oligonucleotide and the corresponding extended region of the barcode label. The obtained extended barcode label comprises in the shown embodiment the following sequence elements: primer sequence (1), B.sub.S, UMI, adapter sequence (1), B.sub.T and primer sequence (2). The reverse complement of the extended barcode label, if provided upon extension, may be removed prior to the amplification step which is performed in step (bb) (see
[0660]
[0661]
[0662]
[0663] After step aa), an amplification step is performed in (bb), wherein a primer or primer combination is used, which comprises the barcode sequence B.sub.P. The amplification reaction is preferably performed in a compartment that does not comprise the cell-laden matrix. If a primer pair is used as illustrated in
[0664]
[0669]
[0674] The presented microfabricated geometry can be e.g. used to accomplish the disclosed methods. In particular, the microfabricated geometry can be used to position for example one-cell-laden hydrogel matrix and a capture matrix in proximity within one compartment. In addition, said microfabricated geometry enables the removal of the capture matrix while the cell-laden matrix remains within its position. One advantage of the presented microfabricated geometry is its compatibility with an array arrangement. Thus, multiple microfabricated geometries can be connected to generate an addressable n×m array containing at least one cell-laden matrix and a capture matrix at each position (n|m) of said array and while still being capable of transferring capture and/or cell-laden matrices located at a defined position. Matrices such as capture matrices or cell-laden matrices can be delivered to the microfabricated geometry within a droplet that is located within a fluid that is immiscible with an aqueous fluid. Said fluid can be an oil such as fluorinated oil (e.g. HFE-7500). If matrices are provided within a droplet, the matrix formation may not have been started, may be ongoing or may be finished (droplet contains a fully polymerized/gelled matrix). In addition, fully polymerized/gelled matrices located within an aqueous phase may be delivered to the microfabricated geometry. For example, capture matrices may be formed prior to the addition to the cell culture device enabling a detailed quality control of the capture matrices using various characterization methods.
[0675]
[0680] In terms of the current disclosure, the generic trapping structure 33 is adapted to position at least two particles such as a cell-laden matrix and a capture matrix. A detailed description of such a positioner is given in
[0681]
[0682]
[0683]
[0684]
[0685]
[0686]
[0691]
[0692]
[0693]
[0694] It is furthermore referred to following Figures of PCT/EP2018/074526, which are including the corresponding figure description herein incorporated by reference: [0695]
[0699]
[0700]
EXAMPLES
[0701] In the following examples, materials and methods of the present invention are provided. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this invention in any manner.
I. General Method Steps
[0702] Cell Encapsulation and Matrix Positioning
[0703] A single cell or multiple cells are encapsulated within a matrix to provide a cell-laden matrix. The matrix material is preferably provided by a hydrogel. Encapsulation into the matrix might be done using techniques such as droplet formation using flow focusing geometries or droplet on demand systems with corresponding sorting mechanisms, subsequent hydrogel formation and demulsification of cell-laden hydrogel matrices located within droplets. A suitable encapsulation method for a particle, here at least one cell, is described in detail in PCT/EP2018/074526, herein incorporated by reference. The described methods inter alia allow to center the cell within a hydrogel bead, thereby providing a cell-laden matrix. Droplet and hydrogel matrix size may be selected in embodiments from a range of 1 μm to 1000 μm, preferably 5 μm to 500 μm, more preferably 30 μm to 200 μm. Suitable ranges were also described elsewhere herein.
[0704] The provided cell-laden matrix is then positioned within a compartment of the cell culture device. The compartment of the cell culture device comprising the cell-laden matrix may have one or more of the following characteristics: [0705] i. it can be selectively opened and closed using microfabricated valves, such as Quake Valves or vertical membrane valves as described in PCT/US2000/017740 or preferably PCT/EP2018/074526, respectively; [0706] ii. it comprises a positioning mean which can be a microfabricated geometry for positioning or immobilizing matrices (e.g. cell-laden matrix and/or capture matrix); and/or [0707] iii. it comprises a microfabricated geometry for removing one or more matrices while one or more other matrices remain within their position. This might be achieved by a valve arrangement adapted to provide fluid passing through a positioning mean (e.g. RFCP geometry as discussed above and as disclosed in PCT/EP2018/074526).
[0708] A capture matrix comprising capture molecules (e.g. immobilized antibodies) having a specificity against a defined biomolecule of interest (e.g. target analytes such as cytokines, chemokines, TNF or interleukins) is provided and positioned next to or in close proximity to the cell-laden matrix (e.g. preferably within the same compartment of a cell culture device, preferably a microfabricated compartment of a cell culture device) so that biomolecules of interest that are released, e.g. secreted, from the at least one cell may diffuse towards the capture matrix so that the capture molecules of the capture matrix can bind and thus capture the biomolecules of interest. Each microfabricated compartment contains a pre-defined number of matrices. In a particular embodiment, a microfabricated compartment comprises exactly one cell-laden matrix and one capture matrix. The distance between the capture matrix and the cell-laden matrix might be between 0 μm (the hydrogel matrices are in direct contact) to 100 μm or more. The positioning of a pre-defined number of different matrices might be achieved using a position mean such as a hydrodynamic trapping structure, preferably a microfabricated geometry for matrix immobilization (as disclosed herein and in PCT/EP2018/074526, herein incorporated by reference).
[0709] Generation of Compartments
[0710] An isolated compartment with a defined volume may be created by selectively closing/isolating the compartment e.g. by actuating corresponding microfabricated valves and/or exchanging the first fluid (e.g. aqueous phase) against a second fluid (which may be a phase immiscible with water, such as an oil phase, preferably a fluorinated oil; also referred to as biphasic compartment generation as described in PCT/EP2018/074526 and PCT/EP2018/074527), whereby the reaction volume is reduced. At this step the capture matrix and the cell-laden matrix are located within the same hydrophilic reaction volume. The reaction volume may be closed by valve actuation, whereby an isolated compartment is generated. In a particular embodiment, the isolated and closed microfabricated compartment has a volume in the range of 1 nL to 500 nL, preferably 10 nL to 50 nL.
[0711] In another advantageous embodiment, the reaction volume can be further reduced by using an alternating biphasic compartment generation described in the present disclosure. Thus, in one embodiment the aqueous phase surrounding the positioned hydrogel matrices might be exchanged by an immiscible fluid such as a fluorinated oil (e.g. HFE-7500) thereby reducing the volume compartment to a volume that approximately corresponds to the volume of the trapped matrices. In one embodiment, the reduced volume of the aqueous phase containing the hydrogel matrices might be in the range of 0.05 nL to 10 nL, preferably 0.4 nL to 0.6 nL.
[0712] Incubation of the Cell-Laden Matrix
[0713] The cell-laden matrix is incubated for a defined time period (e.g. 1 h, 2 h or more). The cell-laden matrix within the compartment is provided in a surrounding/under conditions so that cell(s) located within the matrix may release, e.g. secrete, one or more biomolecules of interest. The biomolecules of interest diffuse to the neighboring capture matrix where they are bound by the immobilized capture molecules having the corresponding specificity towards the released biomolecule of interest. In case an alternating biphasic compartment was generated, the biomolecules of interest may remain within the aqueous phase comprised in the cell-laden matrix, wherein the provided a capture matrix also comprises an aqueous phase, which can become available for diffusion of biomolecules of interest.
[0714] Intermediate Steps Such as Washing Steps
[0715] Washing steps may be performed at any time point throughout the method according to the present disclosure.
[0716] The fixed matrices may optionally be washed with a washing buffer such as PBS to remove unbound biomolecules of interest by perfusing the compartment of the cell culture device.
[0717] According to one embodiment, the isolated compartment is again opened (e.g. by actuating a microfabricated valve). If an aqueous phase surrounding the matrices has been replaced by an oil phase (e.g. HFE-7500), the oil phase may again be replaced by an aqueous phase. This is done by perfusing the microfabricated system with an aqueous phase such as PBS. This procedure is very efficient, as the same buffer can be perfused through all compartments.
[0718] Adding One or More Types of Detection Molecules
[0719] The capture matrix is then contacted with one or more types of detection molecules. The compartment comprising the capture matrix may be in one embodiment perfused with a solution containing one or more types of detection molecules (e.g. with an adjustable concentration) for a defined time period. The detection molecules bind to their captured biomolecules of interest. The detection molecules are associated with a barcode label which comprises at least a barcode sequence (B.sub.s) indicating the biomolecule of interest specificity of the detection molecule. Conjugated detection molecules comprising a barcode sequence for their specificity are commercially available (e.g. from Biogen) and may be used in conjunction with the present invention.
[0720] After adding and incubating the one or more types of detection molecules with the capture matrix, a washing step may be performed (e.g. with PBS) to remove unbound detection molecules.
[0721] The capture matrix with the bound biomolecules of interest may also be transferred to a separate device prior to adding the detection molecules.
[0722] One or more sequence elements may be added to the barcode label, such as a barcode sequence B.sub.T, an UMI sequence for quantification, and/or a barcode sequence B.sub.P, and/or an adapter sequence (AS) for a sequencing platform. As disclosed herein, numerous embodiments exist to introduce these sequence elements and to thereby generate a sequenceable reaction product that comprises one or more of these additional sequence elements.
[0723] Optionally, Adding an Oligonucleotide
[0724] As was described e.g. in detail in conjunction with the above Figures, after addition of the one or more types of detection molecules and optionally washing the capture matrix, an oligonucleotide may be added to extend the barcode label. Suitable embodiments for the oligonucleotide are described in detail elsewhere herein. Such oligonucleotide may comprise e.g. a barcode sequence B.sub.T, an UMI sequence and/or a barcode sequence B.sub.P.
[0725] In one embodiment, the oligonucleotide is capable of hybridizing to the barcode label (also referred to herein as adaptor barcode oligonucleotide). To allow primer extension, the required reagents (e.g. polymerase, dNTPs etc.) may be added after the oligonucleotide was hybridized or the reagents may added, e.g. perfused, into the compartment, together with the oligonucleotide in case a microfabricated device as described herein is used. Conditions are provided to allow extension of the barcode label using the oligonucleotide as template, whereby an extended barcode label is obtained. The polymerase extension reaction can be conducted within in the compartment. Alternatively, the capture matrix can be transported to another position (compartment) of the cell culture device, or a different device, before performing the polymerase extension reaction.
[0726] In an alternative, however less preferred embodiment, the oligonucleotide may be ligated to the barcode label to provide an extended barcode label. Suitable reaction conditions are provided (e.g. ligase, ligase buffer) to allow ligation.
[0727] Transfer and Collection of the Capture Matrix
[0728] The capture matrix (present at a particular position of the cell culture device, e.g. a particular position of an array of positions; and transferred at a pre-defined time point t) comprising the binding complexes of the one or more types of capture molecules, one or more bound biomolecules of interest, and the bound detection molecules may be removed and transferred to a different compartment (position (m, n) being the position of the (preferably microfabricated) compartment, in which the capture matrix (and the corresponding cell-laden matrix) has/have been incubated, t, being the time point at which the capture matrix was removed from the (microfabricated) compartment and transferred e.g. into another format). Therefore, a reverse flow cherry picking mechanism may be used as described in the disclosure of PCT/EP2018/074526, which is herein incorporated by reference, to transfer the capture matrix to a pre-defined collection position (wherein the position information (e.g. compartment position (m, n)) may be maintained by the particular collection position, wherein the collection position for different compartment positions (m, n) may be different) at a pre-defined time point t.sub.x The collection position may e.g. be the well of another format such as a 1536 well plate. The cell-laden matrix can remain in its original position (e.g. inside the microfabricated compartment). According to a particular example, the cell-laden matrix may be trapped in its original position by a microfabricated geometry for matrix immobilization. Also the capture matrix may be trapped by such said microfabricated geometry. The removal of the capture matrix may advantageously be achieved by selectively changing the direction and amount of a fluid by a valve arrangement (also referred to as RFCP mechanism). Such a valve arrangement is described above and the disclosure also applies here. Furthermore such a valve arrangement is disclosed in PCT/EP2018/074526, which is herein incorporated by reference. Such a procedure can be advantageously performed according to the present disclosure, in particular in conjunction with the preferred microfabricated cell culture device.
[0729] After removal of the capture matrix from the proximity of the cell-laden matrix (e.g. removal of the capture matrix from the microfabricated geometry for matrix immobilization), another capture matrix may be added (e.g. to the free position of the microfabricated geometry for matrix immobilization next to the cell-laden matrix). This can be advantageously achieved by the valve arrangement disclosed above (also referred to as the RFCP mechanism). The capture matrix may be added directly or after a predetermined time interval. Hence, the steps described above, starting with the matrix positioning in proximity to the cell-laden matrix may be repeated one or more times. Thereby, information about the released biomolecules of interest at the different time-points is collected and provided in form of a sequenceable reaction product. The method allows to generate a time-resolved profile of released biomolecules of interest.
[0730] Amplification Reaction
[0731] After collecting the desired number of capture matrices from one or more time points or one time-point and numerous positions, an amplification reaction is preferably performed to generate multiple copies of the optionally extended barcode label. As is described herein, one or more sequence elements may be added with the primer or the primer combination that is used for amplification, such as a barcode sequence B.sub.P, a barcode sequence B.sub.T, and/or an adapter sequence (AS) for a sequencing platform. If an UMI sequence is used for quantification, it is introduced prior to amplification. According to one embodiment, a forward primer (e.g. oligonucleotide P-fwd) and a reverse primer (e.g. oligonucleotide T-rev) is used. As is disclosed herein, one or both of the primers of such primer pair may comprise one or more of the sequence elements B.sub.P, B.sub.T, and/or AS.
[0732] The amplification may be a polymerase extension reaction with a single primer (performing repeated cycles of primer extension) or a PCR reaction using a primer pair.
[0733] The amplification reaction using one or more (optionally extended) barcode labels as template is preferably performed within a collection well of a device, such as a well-plate. A LightCycler® 1536 Multiwell Plate and a LightCycler® 1536 Instrument from LifeScience may be e.g. used. As is disclosed herein, an amplification reaction may be e.g. performed in a single collection well using as template the (optionally extended) barcode labels from [0734] a capture matrix obtained at a single time point from at least one cell-laden matrix located in a single compartment; [0735] a plurality of capture matrices obtained at two or more time points from at least one cell-laden matrix located in a single compartment, wherein preferably the barcode B.sub.T is introduced into the (optionally extended) barcode label prior to performing the amplification reaction; [0736] a plurality of capture matrices obtained from a plurality of cell-laden matrices located in a plurality of different compartments at one or more time points, wherein preferably the barcode B.sub.P is introduced into the (optionally extended) barcode label prior to performing the amplification reaction. If the capture matrices were obtained at two or more time points, it is furthermore preferred to also introduce the barcode B.sub.T into the (optionally extended) barcode label prior to performing the amplification reaction.
[0737] Pooling and Sequencing
[0738] After the amplification reaction within each collection position, an aliquot of the generated sequenceable reaction products can be taken from each collection position (e.g. well) and various aliquots may be pooled within a reaction tube. Pooling is possible, as the sequence elements comprised in the sequenceable reaction products allows to identify and correlate each sequenced reaction product e.g. to the original cell-laden matrix and/or time point. The concentration of the pooled sample may be determined (e.g. by using a UV-Vis Spectrophotometer) and adjusted to be compatible with current sequencing procedures. Afterwards the adapted and pooled sample can be sequenced (e.g. by NGS).
[0739] Sequencing Analysis
[0740] The sequencing process will provide the sequencing data for each barcode label within the generated sequenceable reaction products (e.g. barcode library). The sample containing the pooled aliquots from all collection positions contains different barcode labels comprising the specificity information, as well as e.g. the time information, the position information (n|m), as well as the quantity information indicated by a unique molecular identifier. Thus, based on the sequencing data, an analysis algorithm can be employed to extract the mentioned information and to determine the concentration of the biomolecule of interest. In one embodiment, the following algorithm is used: [0741] 1. Identify all barcode sequence that indicated the information about the position of the compartment (position (n, m)) [0742] 2. From said barcode sequence of step 1, identify all barcode sequence that comprises the barcode sequence indicating the time information (e.g. for different time points t.sub.1, t.sub.2, . . . t.sub.x) [0743] 3. From the previously identified barcode sequence of step 2, identify all barcode sequences comprising the barcode sequence indicating the specificity of the detection molecule and thus the biomolecule of interest (B.sub.S1, B.sub.S2, . . . B.sub.Sz) to be analysed (e.g. TNF-alpha or II-6) [0744] 4. From the identified barcode sequence of step 3, count the number of UMIs that are present. This number represents the final concentration (i.e. number) of detected detection molecules at a certain time point. It is assumed, that the binding affinity of the used detection molecules is such that this number is equal to the number of bound biomolecules of interest bound to the capture molecules. Thus with step 4, the concentration of the biomolecule of interest at a certain time point (at a certain position) can be determined.
[0745] Steps 1 to 4 may be repeated if required until the concentration of all biomolecules of interest for all time points for all positions is determined. An illustration of the corresponding data gained with the disclosed method is shown in a more general form in
II. Example 1
[0746] According to Example 1, a method is provided for acquiring a time-resolved profile of one or more biomolecule of interest released by single or multiple cells that are provided in a matrix, preferably a three-dimensional hydrogel matrix. An overview of the process steps of Example 1 is illustrated in
[0747] According to Example 1, one or more types of detection molecules are provided comprising a barcode label comprising following sequence elements: [0748] specificity information (B.sub.S), [0749] primer sequence (1), [0750] adaptor sequence (1), and [0751] a cleavable linker are associated during or after production of the detection molecule (e.g. commercially available antibodies).
[0752] Furthermore, barcode sequences for quantity-, time- and position-information can be added within a collection position (e.g collection well). Sample preparation and handling of capture matrices is performed on a cell culture device, preferably a microfabricated cell culture device. Said microfabricated cell culture device important to the present disclosure as it allows to combine different sequence elements within one oligonucleotide that can be sequenced.
[0753] The method according to example 1 comprises the steps described above in the section about the general method steps. Example 1 differs in comparison to the general method steps in following steps:
[0754] Adding the One or More Types of Detection Molecules
[0755] Adding the one or more types of detection molecules as described above, wherein the detection molecule comprises a barcode label comprising the following elements: [0756] a photo-cleavable linker; [0757] a primer sequence (1) for performing a polymerase chain reaction; [0758] a barcode sequence B.sub.S indicating the specificity of the detection molecule; [0759] an adaptor sequence (1).
[0760] Optionally, Adding an Oligonucleotide to the Capture Matrix in the Compartment
[0761] This step is not performed in Example 1.
[0762] Addition of Information to the Barcode Label
[0763] After collecting the desired number of capture matrices from one or more time points or one time-point and positions, the quantity- (UMI) time- and position-information can be added to the collected detection molecules, in particular the barcode label:
[0764] An oligonucleotide containing an UMI sequence is added to the barcode label encoding the detection molecule specificity by using established methods from molecular biology well known by the person skilled in the art. For example, a polymerization extension reaction or a ligation reaction can be used to transfer the information of oligonucleotide to the barcode label. Afterwards, the barcode sequence indicating the time information and the barcode sequence indicating the position information can be added by using a PCR reaction. An illustration of the process is depicted in
[0765] A polymerase extension/elongation and/or amplification reaction within each collection position (e.g. using a LightCycler® 1536 Multiwell Plate and a LightCycler® 1536 Instrument from LifeScience) whereas the forward primer (oligonucleotide P-fwd) contains a barcode sequence indicating the position information (B.sub.p) and the reverse primer (e.g. oligonucleotide T-rev) contains a barcode sequence representing the time information (B.sub.T) or vice versa to generate an exemplary barcode label (e.g. Oligo-P-Ab-U-T) comprising: [0766] a. the barcode sequence provided by the detection molecule (see above) [0767] b. a unique molecular identifier (UMI) [0768] c. a barcode indicating time information (B.sub.T) [0769] d. a barcode indicating position information for the compartment (B.sub.P) [0770] e. optionally, two sequences complementary to commercially available sequencing primers and adaptors from sequencing companies such as 10× Genomics, Oxford Nanopore, Pacific Biosciences, QIAGEN, Agilent Technologies and Illumina.
[0771] In one embodiment, each well of an exemplary 1536 well plate contains one unique primer combination (e.g. pair of reverse and forward primer). For example, the well A1 contains a reverse primer that comprises a barcode sequence B.sub.T for the time-point t.sub.j and a forward primer that comprises a barcode sequence B.sub.P1 for indicating the position of the compartment (at position (m, n).sub.1) from which the capture matrix was released (position information). The well A2 might contain a reverse primer that comprises a barcode sequence B.sub.T2 for the time-point t.sub.2 and a forward primer that comprises a barcode sequence B.sub.P1 for identifying the position (m, n).sub.1. The well B1 might contain a reverse primer that comprises a barcode sequence B.sub.T1 for the time-point t, and a forward primer that comprises a barcode sequence B.sub.P2 for indicating the position (m, n).sub.2. Thus, for/positions and x time points the needed number of different primers is: n.sub.primer=I*x. This number corresponds to the number of required wells n.sub.wells=n.sub.primer. In one advantageous embodiment, the wells are pre-loaded with lyophilized components necessary for performing the PCR (e.g. by using hot-start PCR) prior to the addition of a capture matrices.
[0772] The described above embodiment has several advantages: First, it enables the analysis of biomolecules that have been released from single cells, cell pairs and/or small cell colonies located within a 3D microenvironment in a dynamic, time-lapse manner. Second, due to the removal of the capture matrix containing the bound biomolecules of interest, the dynamic range of the detection system is large. For example, if only one capture matrix is used for the whole culture time, the capture molecules might be saturated with released biomolecules of interest within minutes to hours resulting in a limited dynamic measurement range. By using multiple capture matrices capturing only the biomolecules of interest released within a defined period, the dynamic range is increased. Third, the reduction of the reaction volume increases significantly the sensitivity of the detection mechanism as the concentration of the biomolecule of interest is higher due to the small volume reduction. Because barcode labels conjugated to detection molecules (preferably antibodies) permit a nearly unlimited number of molecular targets, analytical multiplexing capability is nearly unlimited.
[0773] In addition, the disclosed method offers the following advantages: [0774] The method can be adapted for the detection of any biomolecule of interest, in particular protein, for which a corresponding binding molecule (i.e. detection molecules) such as an antibody is available [0775] The method provides exponential signal amplification due to the use of a polymerase chain reaction (PCR) or polymerase extension reaction which theoretically enables detection of single molecules [0776] Extremely low limit of detection (pg−fg) [0777] Suitable for small sample volumes, in particular for handling of a single cell [0778] Compatible with complex samples [0779] Fewer incubation steps than an ELISA, improved assay reproducibility [0780] Rapid time to results for whole secretome profiles [0781] Wider dynamic range than an ELISA [0782] Highly capable of multiplexing
III. Example 2
[0783] An overview of the process steps of Example 2 is illustrated in
[0784] According to Example 2, one or more types of detection molecules are provided comprising a barcode label comprising following information: [0785] a barcode sequence (B.sub.S), [0786] a barcode sequences for quantity information (UMI), [0787] and the cleavable linker are added during antibody production (commercially available)
[0788] Oligonucleotide sequences for time information and position information are added within a collection well. Antigen binding, washing and handling of capture matrices is performed on a microfabricated cell culture device. Said microfabricated cell culture device is advantageous, as it enables to combine all different information in one oligonucleotide.
[0789] The method according to Example 2 comprises the steps described above in the section about the general method steps. Example 2 differs in comparison to the general method steps in following steps:
[0790] Adding the One or More Types of Detection Molecules
[0791] Adding the one or more types of detection molecules as described above, wherein the detection molecule comprises a barcode label comprising the following elements: [0792] a photo-cleavable linker; [0793] a primer sequence (1) for performing a polymerase chain reaction; [0794] a barcode sequence B.sub.S indicating the specificity of the detection molecule; [0795] a barcode sequences for quantity information (UMI); and [0796] a primer sequence (2) for performing a polymerase chain reaction.
[0797] According to Example 2, the quantity information (UMI sequence) is part of the barcode label bound to the one or more types of detection molecules. To this end, the capture matrix containing one or more types of capture molecules, bound biomolecules of interest and one or more types of detection molecules labeled with barcode labels encode the detection molecules specificity as well as a UMI sequence is transferred into a collection position (e.g. well).
[0798] Conjugated detection molecules having a barcode sequence for their specificity are commercially available (e.g. from Biogen) and can be easily modified with UMI sequences by a skilled person of the art to add the mentioned elements. Degenerate synthesis of oligonucleotides might be used for UMI synthesis.
[0799] Optionally, Adding an Oligonucleotide to the Capture Matrix in the Compartment
[0800] This step is not performed in Example 2.
[0801] Addition of Information to the Barcode Label
[0802] A PCR reaction within each collection well (e.g. using a LightCycler® 1536 Multiwell Plate and a LightCycler® 1536 Instrument from LifeScience) using a primer combination is performed, wherein the forward primer (oligonucleotide P-fwd) contains a barcode sequence representing the position information (Bp) and the reverse primer (oligonucleotide T-rev) contains a barcode sequence representing the time information (BT) or vice versa to generate an exemplary sequenceable reaction product (e.g. Oligo-P-Ab-U-T) comprising: [0803] a) the barcode label provided by the one or more types of detection molecules (e.g. Oligo-Ab-U), [0804] b) a time-point specific nucleotide sequence (B.sub.T), [0805] c) a position specific nucleotide sequence (B.sub.P), [0806] d) adapter sequences (e.g. two sequences complementary to commercially available sequencing primers and adaptors from sequencing companies such as 10× Genomics, Oxford Nanopore, Pacific Biosciences, QIAGEN, Agilent Technologies and Illumina.
[0807] The direct incorporation of the UMI into the detection molecule conjugated barcode label eliminates the need for a primer elongation by reverse transcriptase reactions.
IV. Example 3
[0808] An overview of the process steps of Example 3 is illustrated in
[0809] According to Example 3, one or more types of detection molecules are provided comprising a barcode label comprising following information: [0810] a barcode sequence (B.sub.S), [0811] a barcode sequences for quantity information (UMI),
[0812] Time information is added within compartment of the cell culture device containing a capture matrix and cell-laden matrix. Position information is added within the collection well. Sample preparation and handling of the capture matrix is performed utilizing a microfabricated cell culture device. Said microfabricated cell culture device is advantageous as it enables to combine all different information within one oligonucleotide that can be sequenced.
[0813] The method according to Example 3 comprises the steps described above in the section about the general method steps. Example 3 differs in comparison to the general method steps in following steps
[0814] Adding the One or More Types of Detection Molecules
[0815] The addition of one or more types of detection molecules is performed as described in Example 2.
[0816] Optionally, Adding an Oligonucleotide to the Capture Matrix in the Compartment
[0817] The addition of time information is done by performing an extension of the barcode label bound to the one or more types of detection molecules within the compartment of the cell culture device. After the incubation step and binding, of the biomolecules of interest the compartment containing the capture matrix as well as the cell-laden matrix is perfused with a solution that contains an oligonucleotide with the following elements: [0818] a. a barcode sequence B.sub.T, indicating a time information (e.g. time-point specific sequence) [0819] b. an adapter sequence (1) for binding to the barcode label [0820] c. a reverse primer binding sequence (2).sub.R
[0821] In one embodiment, the solution containing the oligonucleotide might be a hybridization buffer. Due to the perfusion with the hybridization solution, the oligonucleotide binds to the barcode label (that is coupled to the one or more types of detection molecules) via the adaptor sequence (1). Afterwards, unbound oligonucleotides are washed away by perfusion with washing buffer (e.g. PBS). In a next step, the matrices are perfused with a solution containing a DNA-Polymerase such as IsoPol™ DNA Polymerase (ArcticZymes). Thus, the oligonucleotide is extended and the sequence is added to the barcode label (generating and extended barcode label). The extended barcode label contains now the following elements: [0822] a) a photo-cleavable linker, [0823] b) a primer for a polymerase chain reaction (primer sequence (1)), [0824] c) a barcode sequence B.sub.S indicating the specificity of the detection molecule (e.g. an antigen specific sequence (B.sub.S)), [0825] d) a unique molecular identifier (UMI), [0826] e) an adaptor sequence (1), [0827] f) a barcode sequence B.sub.T indicating a time information (e.g. a time-point specific sequence), and [0828] g) a primer sequence (2).
[0829] Addition of Information to the Barcode Label
[0830] Transferring the capture matrix from the compartment (position (m, n)) that contains the one or more types of capture molecules, bound biomolecules of interest and the barcoded one or more types of detection molecule to a pre-defined well (corresponding well to position (m, n)) of another format such as a 1536 well plate. In a preferred embodiment, this is done using the reverse flow cherry picking mechanism as disclosed. At this step, the detection molecules have coupled an extended barcode label that contains the quantity information, the specificity information as well as the time information. The matrix containing the cell(s) remains within its position. As the time information is added when the capture matrix is still positioned within said compartment of the cell culture device, the number of needed wells for generating the sequenceable reaction product is reduced from n.sub.well=I*x to n.sub.well=I.
[0831] After collecting all detection beads from different time points and positions, the position information is added to the collected extended barcode labels that are coupled to one or more types of detection molecules. For example, this is be done by performing a PCR reaction within each collection well whereas the forward and/or reverse primer (here primer combination) might contain a barcode representing the position information.
V. Example 4
[0832] An overview of the process steps of Example 4 is illustrated in
[0833] According to Example 4, one or more types of detection molecules are provided comprising a barcode label comprising following information:
[0834] Antigen-specificity information (B.sub.S).
[0835] Time and quantity information (UMI) is added within compartment of the cell culture device containing amplification matrix and cell-laden matrix.
[0836] In another advantageous embodiment, the time information as well as the quantity information is added to the barcode label bound to the one or more type of detection molecules within the compartment. To this end a oligonucleotide contains a barcode sequence indicating a time information B.sub.T as well as a quantity information (UMI). An advantage is the reduced UMI library size due to combination of UMIs with B.sub.T.
[0837] Apart from the difference above, the method according to Example 4 comprises the steps described above in the section about the general method steps.
VI. Example 5
[0838] An overview of the process steps of Example 5 is illustrated in
[0839] According to Example 5, one or more types of detection molecules are provided comprising a barcode label comprising following information: [0840] Antigen-specificity (B.sub.S)/Time(B.sub.T)/Quantity (UMI) information is added during antibody production
[0841] In another advantageous embodiment, the barcode label bound to the one or more types of detection molecule contains the specificity, the quantity and the time information thereby reducing the number of processing steps. Thus, after incubating the capture matrix and the cell-laden matrix (or matrices) and subsequent washing, the capture matrices are perfused with a solution containing one or more types of detection molecules that are labeled with the barcode label containing the specificity, quantity and time information. The capture matrices are finally transferred to a collection well where the position information is added for example by using a PCR.
[0842] Apart from the difference above, the method according to Example 5 comprises the steps described above in the section about the general method steps.
VII. Example 6
[0843] The core process steps of Example 6 are illustrated in
[0856] It is noted that the incubation period may be selected by the skilled person in view of the cells comprised in the cell-laden matrix and the biomolecule(s) of interest. In embodiments, the incubation period is selected from the range of 1 h to 72 h, such as 4 h to 72 h. A shorter incubation period (e.g. 1 h to 24 h) may be selected for microbiological applications. For instance, a shorter incubation period may be selected for a prokaryotic cell, such as a bacterial cell, which can be comprised in the cell-laden matrix as disclosed herein. A longer incubation period (e.g. 4 h to 72 h) may be selected for other applications. For instance, a longer incubation period may be selected for a eukaryotic cell, such an animal cell, which can be comprised in the cell-laden matrix.
[0857] The method may also comprises one or more cycles of incubation of the cell-laden matrix to allow release on the one or more biomolecules of interest and capture matrix addition in each cycle as discussed above. The repeated incubation and binding can be performed multiple times, e.g. ≥two times, ≥three times, ≥four times, or ≥five times. Suitable time intervals between cycles can be selected by the skilled person. In embodiments, the time interval between cycles is selected from ≥10 min, ≥20 min, ≥30 min, 1 h, ≥2 h, ≥3 h, ≥4 h, 5 h or more, up to days 1 d, 2 d or several days, preferably selected from the range of 30-130 min.