Extracellular Matrix Gels, and Organoid Cultures Comprising the Same
20220389375 · 2022-12-08
Assignee
Inventors
- Paolo DE COPPI (London, GB)
- Nicola ELVASSORE (London, GB)
- Giovanni Giuseppe GIOBBE (London, GB)
- Claire CROWLEY (London, GB)
Cpc classification
C12N5/0062
CHEMISTRY; METALLURGY
A61K35/38
HUMAN NECESSITIES
C12N2533/90
CHEMISTRY; METALLURGY
C12N5/0677
CHEMISTRY; METALLURGY
C12N5/0671
CHEMISTRY; METALLURGY
A61K35/12
HUMAN NECESSITIES
International classification
C12N5/00
CHEMISTRY; METALLURGY
A61K35/38
HUMAN NECESSITIES
Abstract
The invention concerns novel methods and materials for preparing extracellular matrix (ECM) powder pre-gel and gel solutions, for example for use in organoid culture. The ECM gels demonstrate excellent physiological and mechanical properties while having the proteomic signature of endoderm tissue with specific enrichment of key ECM proteins relevant to organoid formation.
Claims
1. A method of preparing an extracellular matrix powder pre-gel solution (“ECM pre-gel”), the method comprising: (a) providing decellularised tissue; (b) processing the decellularised tissue to derive ECM powder from said tissue; and (c) digesting the powder at a concentration of 1 mg/mL to 8 mg/mL in a proteolytic solution, thereby forming said ECM pre-gel.
2. The method of claim 1, wherein the decellularised tissue is intestinal tissue, optionally small intestinal tissue.
3. The method of claim 2, wherein the small intestinal tissue is the mucosal/submucosal layers of the small intestine.
4. The method of any of the above claims, wherein the powder is digested at a concentration of 2 mg/mL to 8 mg/mL, 2 mg/mL to 6 mg/mL, or at a concentration of 2 mg/mL to 4 mg/mL.
5. The method of any of the above claims, wherein, prior to step (c), the ECM powder is: sterilized by gamma radiation; and/or the decellularised tissue is washed in Milli-Q water and then washed with DNase.
6. A method of preparing an extracellular matrix powder gel solution (“ECM gel”) prepared by the following steps: (i) performing the method of any one of claims 1 to 5, (ii) neutralising the ECM pre-gel to form a gel solution with a pH of between 6.8 to 7.7, thereby forming said ECM gel.
7. The method of claim 6 wherein, prior to step (ii), the ECM pre-gel is centrifuged, and precipitated undigested pellets of decellularized tissue, that result from the centrifugation step, are discarded.
8. The method of claim 6 or claim 7, wherein, in step (ii), the ECM pre-gel is neutralised in Dulbecco's Modified Eagle Medium (DMEM).
9. An ECM pre-gel prepared by the method of any one of claims 1 to 5.
10. An ECM gel prepared by the method of any one of claims 6 to 8.
11. An organoid culture, comprising: organoids, pieces of organoids, or organoid cell pellets; and an ECM gel of claim 10.
12. A method of preparing an organoid culture, comprising mixing: organoids, pieces of organoids, or organoid cell pellets; and an ECM gel of claim 10; thereby forming said culture.
13. The method of claim 12, further comprising aliquoting droplets of the mixture onto a suitable medium, optionally where the suitable medium is a petri dish, and/or optionally wherein the droplets have a volume of 30-40 μL.
14. The culture or method of any one of claims 11-13, wherein the organoids or organoid cell pellets are endoderm-derived organoids or organoid cell pellets.
15. The culture or method of any one of claims 11-14, wherein the endoderm-derived organoids or organoid cell pellets are selected from: organoids or organoid cell pellets of gastric origin; stomach enteroids or enteroid cell pellets; pediatric stomach enteroids or enteroid cell pellets; ductal (cholangiocyte) organoids or organoid cell pellets; fetal hepatic (hepatocyte) organoids or organoid cell pellets; intestinal stem cells or stem cell pellets; Lgr5+ intestinal stem cells or stem cell pellets; organoid or organoid cell pellets of fetal origin; small intestinal enteroids or enteroid cell pellets; adult cholangiocyte ducts; fetal hepatocyte organoids or organoid cell pellets; human ductal organoids or organoid cell pellets; ductal liver organoids or organoid cell pellets; intestinal organoids or organoid cell pellets; or fetal pancreatic organoids or cell pellets.
16. The culture or method of any one of claims 11-15, wherein the organoids or organoid cell pellets are human or mouse organoids or organoid cell pellets.
17. The culture or method of any one of claims 11-16, wherein the organoid culture can be maintained for more than two passages.
18. The culture or method of any one of claims 11-17, wherein the organoid culture can maintain expression of essential markers after seven days from culture formation.
19. The culture or method of any one of claims 11-18, wherein the organoid culture can be maintained for more than one month from culture formation.
20. The culture or method of any one of claims 11-19, wherein the culture does not comprise Matrigel and/or basement membrane extract.
21. The culture or method of any one of claims 11-20, wherein gene expression in the organoids or organoid cell pellets of the culture is comparable to gene expression in an organoid or organoid cell pellet culture comprising Matrigel.
22. The culture or method of claim 21, wherein the gene is selected from the group consisting of: LGR5, OLFM4, SMOC2, LYZ, BMI1, LRIG1, FABP1, MUC1, MUC3A, MUC5B, EZR, VIL1, MUC12, MUC13, MUC17, MUC20, CHGA, TINAGL1, LTBP4, CRELD1, ECM1, LGALS1, LGALS3, LMAN1, P4HA1, KRT7, KRT8, KRT18, KRT19, EPCAM, SOX9, TACSTD2 (TROP2), ALB, ASGR1, ASGR2, SERPINA1, FABP1, APOA2, ALPI and MUC2.
23. A method of in vivo delivery of an organoid culture to a subject, comprising administering to the subject the culture of claim 11 or claims 14-22, or the culture obtained by the method of any one of claims 12-22.
24. A method of treatment of disease in a subject, comprising administering to the subject the culture of claim 11 or claims 14-22, or the culture obtained by the method of any one of claims 14-22.
25. The method of claim 23 or claim 24, wherein the culture is conducted for 2-4 days prior to administration.
26. The method of any of claims 23-25, wherein the subject is a human subject, a mouse subject, or a mouse model of disease.
27. The method of any of claims 25-26, wherein the organoid is a human fetal pancreatic organoid or small intestinal organoid.
28. The method of any of claims 23-27, wherein organoid organisation is preserved after 2 weeks, after 3 weeks, after 4 weeks, after 5 weeks, after 6 weeks, after 7 weeks, or after 8 weeks from administration.
29. The method of any of claims 23-27, wherein the culture comprises matured organoids after 2 weeks, after 3 weeks, after 4 weeks, after 5 weeks, after 6 weeks, after 7 weeks, or after 8 weeks from administration.
30. The method of any of claims 23-29, wherein gene expression of the organoid is comparable to gene expression in to an organoid culture comprising Matrigel.
31. The culture of claim 11 or claims 14-22, or the culture obtained by the method of any one of claims 12-22, for use in the method of any of claims 23-30.
32. Use of the culture of claim 11 or claims 14-22, or the culture obtained by the method of any one of claims 12-22, in the manufacture of a medicament for the method of any of claims 23-30.
33. An organoid culture comprising: organoids, pieces of organoids or organoid cell pellets; and an extracellular matrix powder gel solution (“ECM gel”) comprising ECM powder at a concentration of between 1 mg/mL and 8 mg/mL.
34. The culture of claim 33, wherein the organoids or organoid cell pellets are endoderm-derived organoids or organoid cell pellets.
35. The culture of claim 33 or claim 34, wherein the endoderm-derived organoids or organoid cell pellets are selected from-organoids or organoid cell pellets of gastric origin; stomach enteroids or enteroid cell pellets; pediatric stomach enteroids or enteroid cell pellets; ductal (cholangiocyte) organoids or organoid cell pellets; fetal hepatic (hepatocyte) organoids or organoid cell pellets; intestinal stem cells or stem cell pellets; Lgr5+ intestinal stem cells or stem cell pellets; organoid or organoid cell pellets of fetal origin; small intestinal enteroids or enteroid cell pellets; adult cholangiocyte ducts; fetal hepatocyte organoids or organoid cell pellets; human ductal organoids or organoid cell pellets; ductal liver organoids or organoid cell pellets; intestinal organoids or organoid cell pellets; or fetal pancreatic organoids or cell pellets.
36. The culture or method of any one of claims 33-35, wherein the organoids or organoid cell pellets are human or mouse organoids or organoid cell pellets.
37. The culture or method of any one of claims 33-36, wherein the organoid culture can be maintained for more than two passages.
38. The culture or method of any one of claims 33-37, wherein the organoid culture can maintain expression of essential markers after seven days from culture formation.
39. The culture or method of any one of claims 33-38, wherein the organoid culture can be maintained for more than one month from culture formation.
40. The culture or method of any one of claims 33-39, wherein the culture does not comprise Matrigel and/or basement membrane extract.
41. An extracellular matrix powder gel solution (“ECM gel”) prepared by the following steps: (a) providing decellularised tissue; (b) processing the decellularised tissue to derive ECM powder from said tissue; (c) digesting the powder at a concentration of 1-20 mg/mL, or optionally 2-20 mg/mL, or optionally 5-15 mg/mL, or optionally 10 mg/mL, in a proteolytic solution, thereby forming an ECM pre-gel; (d) neutralising the ECM pre-gel to form a gel solution with a pH of 6.8 to 7.7, thereby forming said ECM pre-gel; and (e) allowing the ECM pre-gel to gelate; and (f) mixing the ECM pre-gel with synthetic pre-polymer.
42. The ECM gel of claim 41, wherein the synthetic pre-polymer comprises poly-acrylamide.
Description
FIGURES
[0161]
[0162] These figures are discussed in example 1.
[0163]
[0164] These figures are discussed in example 2.
[0165]
[0166] These figures are discussed in example 3.
[0167]
[0168] These figures are discussed in example 4.
[0169]
[0170] These figures are discussed in example 5.
EXAMPLES
[0171] The following examples are provided so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make the compositions of the invention and how to practice the methods of the invention and are not intended to limit the scope of what the inventor regards as his invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.), but some experimental errors and deviation should be accounted for. Unless indicated otherwise, temperature is in degrees C., chemical reactions were performed at atmospheric pressure or transmembrane pressure, as indicated, the term “ambient temperature” refers to approximately 25° C. and “ambient pressure” refers to atmospheric pressure. The invention will be further clarified by the following examples which are intended to be exemplary of the invention.
Example 1—Gelation and Characterization of ECM-Derived Intestinal Hydrogel
[0172] To optimize a GMP-compatible process for ECM gel, a 5 step protocol was designed which includes (i) tissue harvesting; (ii) decellularization; (iii) freeze dry and milling; (iv) gamma-irradiation and digestion; and (v) neutralization (
[0173] It was first verified that ECM powder derived from porcine intestinal tissue successfully formed a hydrogel when following a gelation protocol. ECM powder was digested in pepsin and HCl to form a pre-gel, re-equilibrated to neutral pH and exposed to physiological temperature. The SI ECM decellularization and gelation efficiently preserved the relevant extracellular matrix components including collagens, elastin and still contained glycosaminoglycans (
[0174] Rheological and mechanical properties of 3D environment are relevant to organoids culture. By spectrophotometry the turbidimetric gelation kinetics of the hydrogels were assessed (
TABLE-US-00001 TABLE 1 Gelation characterization of different ECM concentrations Gelation Half-time Lag Period (T.sub.lag) Gelation Rate (S) (T.sub.1/2) ECM gel (min, ±SD) (AU/min, ±SD) (minutes, ±SD) 10 mg/ml 16.39 ± 0.63 0.047 ± 6.33 × 10.sup.−4 26.92 ± 0.78 8 mg/ml 15.96 ± 0.82 0.048 ± 7.59 × 10.sup.−4 26.30 ± 0.59 6 mg/ml 12.79 ± 1.37* 0.059 ± 2.66 × 10.sup.−3 21.25 ± 0.83
However, the ECM powder digested solution (pre-gel) preferably needed to be freshly prepared and kept at +4° C., or stored at −20° C., because, in these conditions, room temperature (1 month) stored pre-gel failed gelation as no sigmoidal curve was observed (
[0175] Rheological characteristics of different ECM gel concentrations and Matrigel were assessed using temperature ramping oscillatory rheology (
Example 2—Intestinal ECM Hydrogel Proteomic Profiling
[0176] To characterize the ECM composition in terms of residual proteomic content after decellularization, a mass spectrometry analysis was performed on the powder form, before pepsin digestion.sup.11. More than 1600 proteins were identified, of which ˜130 were recognized as derived from ECM and 619 from extracellular exosomes (
[0177] Despite the high diversity of identified proteins, most of them contributed for less than 1% of the total amount. Multiple collagen types were among the most abundant, mostly fibrillar (type 1, 2, 3, 5, 6), but also fibril-associated (type 12, 14, 21) and sheet forming (type 4) (
[0178] To verify if the retained complex protein composition of the ECM gels showed similarities with specific human tissues, the ECM proteins quantified in the data were searched on a publicly available map of the human proteome (
[0179] The similarity between the ECM protein composition of the decellularized matrices and the above tissues was also quantitatively investigated by principal component analysis, which showed a higher similarity of the ECM gels composition with tissues of endodermal origin (
Example 3—ECM Hydrogels Support Both Mouse and Human Organoids Cultures
[0180] The possibility of ECM gels to host different endoderm-derived organoids cultures was then demonstrated. Extensive analysis was performed on both human and mouse organoid cultures, from different organs. First, human organoids of gastric origin showed high level of adaptation to the small intestinal ECM gel. The pediatric stomach enteroids maintained the expression of both epithelial (zonula occludens-1, epithelial cadherin and f-actin) and gastric (ezrin and mucin-5AC) markers after 7 days of culture (
[0181] Different endodermal organoids such as human ductal (cholangiocytes) and human fetal hepatic (hepatocytes) organoids from different donors were also explored, and the morphology with two different standard controls such as BME and Matrigel (
[0182] Lgr5+ intestinal stem cells, isolated from the crypts of the mouse small intestine, survived and maintained their phenotype, forming expanding enteroids in the ECM hydrogel over time (
[0183] Importantly, similar features were observed when human pediatric small intestinal organoids were cultured in ECM gels. Live/Dead assay showed a comparable number of live cells to those cultured in Matrigel demonstrating extensive cytocompatibility of ECM hydrogels, with standard viability potential (
[0184] The possibility of ECM gels to host human organoid cultures of fetal origin was then explored. Small intestinal enteroids showed ideal morphology (
[0185] To assess the safety of the ECM gel, the residual presence of galactose-alpha-1,3-galactose (alpha-gal) was analysed. The presence of the antigen with immunofluorescence analyses in the non-decellularized small intestinal mucosa, the intact decellularized tissue, and 6 different batches of the piglet SI-derived ECM gel was screened for. It was possible to assess the presence of the porcine antigen both in the fresh and in the whole decellularized tissue, as already reported.sup.25. On the other hand, the presence of the antigen in any of the 6 analyzed gels was not observed. These results showed the absence of alpha-gal in the final ECM gel, or the extreme dilution of the epitope that could be detected (
[0186] It might be important to modulate the physical and biochemical properties of the ECM gel by adding further hydrogel-forming components. By using a synthetic hydrogel system made of poly-acrylamide, a highly homogeneous hydrogel in which the collagen fibers are uniformly interspersed (
Example 4—ECM Gel Culture Human Organoids Transcriptomic Profile
[0187] To better understand the behavior of human organoids in ECM gel compared to Matrigel, the cultures were further characterized through transcriptomic and functional analyses. 3′ RNA-sequencing was performed on human small intestinal organoids derived from a pediatric donor (
[0188] Of all preserved proteins identified in the decellularized ECM, 109 (˜6% of all DEGs) were also identified as differentially expressed at the transcriptomic level, with 42 up-regulated in ECM and 67 up-regulated in Matrigel. As for the transcripts known to be in the core matrisome.sup.27 (
[0189] These outcomes on human SI organoids outline the strict connection between microenvironment and cell physiology, therefore moving to a native ECM might benefit organoid phenotype. It was also checked by real-time PCR the differential expression of some major intestinal markers observed in the SI RNA-sequencing in
[0190] Moreover, a full set of transcriptomic data on human liver cells is reported. For this, human adult cholangiocyte ducts, and human fetal hepatocyte organoids, previously presented in
[0191] The RNA-seq analysis for the human fetal hepatic organoids highlighted also in this case a distance between ECM gel and BME cultured organoids, as shown in the PCA plot and in the heatmap of the differentially expressed genes (
[0192] Interestingly, the functional analysis on the production of human albumin by both ductal.sup.23 and hepatic.sup.24 organoids showed a comparable secretion in both ECM gel and BME cultures (
Example 5—In Vivo Delivery of ECM Cultured Organoids
[0193] In vivo delivery of cultured human organoids was explored, which remains challenging as it is linked to efficient vascular support, and the possibility of using clinically compatible vectors. For this purpose, human fetal pancreatic organoids were used as a test system. Fetal ducts grown in vitro in ECM gel and Matrigel showed similar morphology (
[0194] In order to evaluate the angiogenic potential of ECM gels, the Chick Chorioallantoic Membrane (CAM) assay was performed (
[0195] To further evaluate the in vivo potential, pancreatic organoids within ECM gels (and Matrigel as control) were also seeded, and transplanted subcutaneously in immunodeficient mice. Cells were then harvested at 2.5 (
[0196] As an additional application of this model, a set of in vivo experiments with small intestinal organoids was performed. To this end, the LGR5-DTR-EGFP mouse model.sup.30 was used. Mouse small intestinal organoids with GFP-reporter crypt stem cells, which could be traced after an in vivo transplant, were derived. These cells were transplanted in ECM gels, into mice back sub-cutaneous pockets. After one month, all 5 ECM gels transplanted were retrieved, which contained matured organoids (
Example 6—Discussion and Materials and Methods
[0197] Discussion
[0198] Disclosed here is the successful development of ECM gels that have the potential to both direct and influence human organoids behavior in vitro and in vivo. This includes directing cell adhesion, survival, proliferation, and differentiation, while also providing a mechanical support to the cells. An ex vivo 3D cell culture support should ideally recapitulate aspects of this native microenvironment and facilitate these functions.sup.31. LGR5+ cells, isolated from the crypts of the intestine are an example of a cell type that favors a 3D environment for ex vivo culture over 2-D.sup.32. A 2D culture, provides an unnatural environment for the cells. In a monolayer culture, only a portion of the cell surface is in contact with ECM and neighboring cells, with the remaining portion exposed to the culture media. This provides a homogeneous supply of nutrients, cytokines and growth factors to this external membrane, which unlikely resemble the dynamic spatial gradient of nutrient supply received in vivo.sup.33.
[0199] Both natural and synthetic hydrogels have been examined for their ability to support organoid culture, each having their own associated advantages and limitations. Recently, synthetic alternatives to Matrigel have been reported.sup.9,10. While synthetic gels have the advantage of being GMP-compliant and reproducible, they are limited by a lack of biological signals provided to the cells. ECM is far more complex and this disclosure demonstrates that this information allowed clustering within the germ layer of derivation. The ECM gels of the disclosure can support the culture not only of intestinal organoids, but also cells derived from other endoderm-derived tissue such as liver, stomach and pancreas.
[0200] Porcine intestine tissue was decellularized using the DET protocol as disclosed herein. Mesentery and the external muscle layer were removed in situ using an in-house established protocol. One cycle of the DET protocol was required to remove nuclei from the scaffold which was confirmed with H&E staining along with a significant reduction in DNA content. Histological analysis confirmed the presence of collagen, elastin and GAGs post gelation. Collagen increase compared to tissue weight is a common feature following decellularization and loss in cytoplasmic compartment. Further characterization highlighted maintenance of the main collagen isoforms.sup.8. Spectrophotometry and rheology experiments confirmed gelation of the ECM hydrogel at all concentrations. Gelation also preserved appropriate stiffness which is fundamental for enteroid formation, survival and differentiation.
[0201] Past works describe how ECM is not only a mere scaffold, but it is an integral determinant of tissue specificity itself. Epithelial and mesenchymal components interact during development to direct tissue morphogenesis and differentiation. The tissue development is not a cell autonomous process, but it is instead instructed by the surrounding environment.sup.34. Extensive proteomic analysis on the decellularized tissue powder further confirmed that the major extracellular matrix components were preserved, such as the main collagen isoforms, but also non-extracellular matrix components, which may play a role in the signal transduction of the organoids cultured in the gel. Small intestinal ECM proteomic profile clusters with the main endoderm-derived organ's ECM profiles. This characteristic suggests that this gel could also be used to support the culture of both mouse and most relevantly human organoids derived from stomach, adult (ducts) and fetal (hepatocytes) liver, adult and fetal small intestinal mucosa, and fetal pancreas. Many exosomal proteins were also preserved within the decellularized matrix, including proteins related to cell adhesion. The metabolomics analysis on the digested powder allowed sub-products of protein degradation and fatty acid residual to be identified, which were expected to be found after cell membrane breakdown during decellularization process.
[0202] Transcriptomic analyses performed on ECM gel cultures showed how human organoids maintain their identities compared to Matrigel cultures, and this is confirmed in human small intestine, liver duct, and hepatic organoids. Interestingly, pediatric small intestinal organoids maintained a higher proportion of crypt stem cells in SI ECM hydrogels compared to slightly more differentiated cells in Matrigel. This may advantageously allow for more efficient propagation of a culture. Nonetheless, all the specific intestinal markers, defining both crypt and villi signature, were expressed in ECM gel compared to Matrigel cultured organoids, underlying the suitability of the ECM to host human cultures.
[0203] On the other hand, ECM specific markers that were detected at the proteomic level in the decellularized ECM were found to be all overexpressed in Matrigel cultured SI organoids. Only few of these were previously reported to be present in Matrigel.sup.28. This observation might be ascribed to the necessity of SI organoids to produce their intestinal extracellular matrix, compared to ECM gel cultured organoids that are already integrating signals from the native small intestinal ECM.
[0204] A translational application of ECM-derived hydrogels is currently hampered by the high variability of the lab-derived products. In our study, to better standardize the process we always used similar age and similar weight (3 kg) piglets of the same pure ‘Petrain’ breed. Each new batch was then tested for ECM digestion quality, gelation quality, stability in culture medium in incubator and cytocompatibility with organoids.
[0205] We furthermore combined the ECM hydrogel of the invention with synthetic molecules, for example photo-polymerizable polyacrylamide to obtain a combined gel suitable for monolayer growth of mouse and human small intestinal organoids. Based on these results it appears other synthetic materials could be used e.g. polyglycolic acid (PGA), polylactic acid (PLA).
[0206] Long term expansion requires stable and consistent cultures. In some embodiments, the cultures displayed comparable outcome during the first 3-4 passages, which would be sufficient for ex vivo cell expansion. Moreover, this did not affect in vivo delivery of the cells, which is ultimately one of the main objectives of the ECM gel.
[0207] In view of GMP-grade production for clinical use, it is important to underline that all the chemicals and reagents utilized during each step of the gel production pipeline are already commercially available at GMP-grade. This list is presented in the following table.
TABLE-US-00002 Good Manufacturing Practice (GMP) Product Pack products Company code size Milli-Q ® Integral ultrapure water Type I, Merck Millipore ZRXQ015WW unlimited 0.22 μm filtered Sodium deoxycholate (SDC) PharmaGrade Merck - Sigma S1827 1 kg manufactured under appropriate controls Aldrich Sodium chloride (NaCl) PharmaGrade, Merck - Sigma RES0926S- 25 kg USP, Manufactured under appropriate GMP Aldrich A705X controls DNase I, recombinant, RNase-free, GMP Roche - 03724751103 4 kU Grade CustomBiotech GMP-certified and virus validated Pepsin, Biofac Pepsin 1 kg pharmaceutical and technical grade Powder - GMP grade Probumin ® Bovine Serum Albumin (BSA) Merck - Sigma 820476 5 kg Biotech Grade Aldrich Hydrochloric acid (HCI) Grade ACS Merck - Sigma 30721-1L-M 1 L reagent, reag. ISO, reag. Ph. Eur. 37% Aldrich
[0208] Importantly, as a further step towards the applicability to a clinical translatable protocol, this disclosure demonstrates the possibility to derive organoids from human biopsies without the use of Matrigel at the first passage after tissue dissociation.
[0209] Further, human organoids cultures of the disclosure can survive in vivo maintaining both structure and signature expression at protein level. Importantly, angiogenesis occurred already at 2.5 weeks post-transplantation in vivo and increased over time with no major differences between ECM gel and Matrigel.
[0210] The in vivo results disclosed highlight the utility of the ECM gel of the disclosure to efficiently deliver cells of both human and animal origin. Moreover, the ECM gel of the disclosure facilitated cell survival of up to 2 months for enteroids derived from different organs, as shown with the fetal pancreatic ductal organoids, and the small intestinal organoids.
[0211] Materials and Methods
[0212] Porcine Intestinal Tissue Collection
[0213] Porcine (Sus scrofa domesticus) small intestinal (SI) mucosal/submucosal layers from the ‘Pietrain’ breed were used. Piglets up to 3 kg in weight were euthanized via blunt trauma once the criteria outlined by the JSR veterinary advisors had been met. Once sacrificed, the animals were transported to the lab via courier and the intestine was harvested immediately on arrival (within 6 hours of euthanasia). The whole small intestine was harvested (duodenum, jejunum, and ileum) and the internal tube was pulled out leaving behind the external layer and mesentery. The retrieved mucosal/submucosal tissue was then extensively cleaned with pressurized water, opened longitudinally, cut into 5 cm or 4 cm or 3 cm or 2 cm or 1 cm pieces and placed in Milli-Q® (Merck Millipore) water overnight at 4° C., on a laboratory rotator, or in a magnetic stirrer or agitator to begin the first step of decellularization. Each batch of ECM was composed of 3 pooled piglets SI.
[0214] Decellularization of Porcine Intestine Tissue
[0215] The detergent-enzymatic treatment (DET) for decellularization, previously established on rat small bowel was optimized for the porcine intestine.sup.20. After the first overnight wash, the tissue was decellularized at 4% sodium deoxycholate (Sigma Aldrich) for 4 hr at room temperature (RT). This was followed by a washing step in Milli-Q water for 24 h at RT, with multiple water changes throughout, and then a step of 2000 kU DNase-I (Sigma Aldrich) in 1M NaCl (Sigma Aldrich) for 3 hr at RT. The tissue was then placed in Milli-Q water and washed for 2 days or 3 days, with multiple water changes. Wash steps are important for removing any cytotoxic residual of sodium deoxycholate. A laboratory rotator or magnetic stirrer was used throughout the decellularization process.
[0216] Gelation Protocol and Cell Inclusion
[0217] The decellularized porcine intestine was freeze dried for 72h (Labconco FreeZone Triad Freeze Dry Systems), milled into a thin powder using a mini mill (Thomas Wiley, mesh 40), sterilized by gamma irradiation (17 kGy for 10h) and stored at −20° C. until further use. For gelation, the ECM powder was digested at 4 or 6 or 8 or 10 mg/ml in pepsin/HCl solution (1 mg/ml in 0.1M HCl) at RT for 72 hours, in constant rotation. For preparing organoid cultures, the ECM powder was digested at a concentration of less than 8 mg/mL. Pre-gel was then centrifuged (200-400 g for 3-5 min) to precipitate and discard eventual undigested particles. Acidic pre-gel solution was commonly used freshly prepared, but it could be stored at 4° C. up to 1 month, or frozen in aliquots at −20° C. for prolonged storage. For cell seeding, while working on ice and immediately prior to use, pre-gel solution was equilibrated to cytocompatible salinity adding 10% 10X PBS for mechanical tests, or 10X DMEM F/12 (Thermo Fisher) for cell culture and neutralized to physiological pH of 7.5 by addition of NaOH 10M and thoroughly mixing, with modification of published protocols.sup.18,19. During these steps, cut-end pipette tips are used, to facilitate dense gel pipetting. Gel was mixed with cell pellets and aliquot in 30-40 μL droplets in Petri dish. Gelation took place in 30 min in the incubator. Organoids were optionally cultured in 4-6 mg/mL ECM gels.
[0218] Tissue Histology
[0219] Tissue samples were taken at random immediately post-harvesting and after each cycle of decellularization. For paraffin embedded sections, samples were fixed in 4% paraformaldehyde solution in PBS for 24 hours at RT, washed in dH.sub.2O, dehydrated in graded alcohol, embedded and cut into 5 μm sections. For frozen sections, samples were snap frozen in liquid nitrogen, placed in OCT and cut into 7 μm sections. For ECM gel and Matrigel® Basement Membrane Matrix Growth Factor Reduced (GFR) (Corning 354230), droplets were fixed in glutaraldehyde 2% for 2 h. After fixing, another PBS wash was followed with 100-150 μL of 2% agarose solution until the droplet was fully covered. The agarose was removed, taking with it the gel droplet and stored in 70% ethanol. The agarose/hydrogel samples were then dehydrated with a series of ethanol washes with increasing concentrations followed by two xylene washes. Samples were embedded in paraffin and cut into 7 μm sections. Tissue slides were stained according to manufacturers' instructions with Hematoxylin and Eosin (H&E) (Thermo Fisher) and Hoechst 33342 (Thermo Fisher) to determine the presence of nuclei and Picrosirius Red (PR), Elastic Van Gieson (EVG) and Alcian Blue (AB) (Thermo Fisher) to assess retention of collagen, elastin and glycosaminoglycans respectively.
[0220] DNA and ECM Quantification
[0221] Tissue samples were taken at random immediately post-harvesting and after decellularization protocol for DNA and ECM components quantification. DNA was quantified using a PureLink Genomic DNA Mini Kit (Thermo Fisher). The final concentration of DNA in the samples was measured using a NanoDrop (model NanoDrop 1000 Spectrophotometer by Thermo Fisher). ECM components were quantified using a QuickZyme Collagen assay kit (QuickZyme Biosciences) to measure the collagen, a Blyscan Sulfated Glycosaminoglycan Assay kit (Biocolor) for the glycosaminoglycans (GAGs) and a Fastin Elastin Assay kit (Biocolor) for elastin, according to manufacturers' instructions.
[0222] Turbidity
[0223] The turbidity of the hydrogels was assessed using spectrophotometry (n≥5). 200 μl of the hydrogel were pipetted into a 96-well plate and absorbance at 450 nm was measured at 37° C. once per min for 1 hr.sup.36. Readings were normalized to a PBS control and then normalized using the calculation below, where NA is the final normalized absorbance, R is the absorbance reading obtained at a given time, R.sub.min is the smallest absorbance value recorded and R.sub.max is the greatest absorbance value. From the data, the half gelation time (t1/2), the gelation rate (S) and the lag time (tlag) was calculated.
[0224] Oscillatory Rheology
[0225] The neutralized gel (3 mL) was placed in between the two plates of the rheometer heated to 37° C. with a gap size of 1 mm, and a sinusoidal stress of constant maximum amplitude of 0.5 Pa applied at a frequency of 1 Hz. The resulting strain was measured for approximately 1 hour 30 minutes.sup.36.
[0226] Oscillatory rheology was performed as a temperature ramping study using a Discovery HR-2 rheometer (TA instruments). 1 mL of the neutralized digested hydrogel was poured onto the preheated steel peltier plate, 4° C. for Matrigel (100% concentration) and for the ECM gel. The 40 mm parallel plate is lowered to a gap of 650-800 μm, or until the gel perfectly fills the gap. The sinusoidal stress of constant 21 maximum amplitude of 50 Pa was applied at frequency of 25 Hz, with a temperature ramp from 22-37° C. for 7.5 minutes, a constant temperature of 37° C. for 45-75 min and finally another temperature ramp from 37-50° C. for a period of 7.5 min. G′ and G″ were measured for the entire period.
[0227] Scanning Electron Microscopy (SEM)
[0228] SEM-images of the cross-section, top and bottom surfaces of the ECM gel were taken to examine surface-topography of the material. All samples were fixed in 2.5% glutaraldehyde (Sigma Aldrich) washed in 0.1 M phosphate buffer (pH 7.4), post fixed with 1% OSO.sub.4 (osmium tetraoxide)/1.5% Potassium Ferrocyanide K.sub.4[Fe(CN).sub.6] in 0.1 M phosphate buffer followed by a dH.sub.2O wash. Specimens were then dehydrated in a graded ethanol-water series to 100% ethanol (50%, 60%, 70%, 80%, 90%, 95%, 100%) and critical point-dried using CO.sub.2.sup.37. The samples were mounted onto aluminum stubs using sticky carbon tabs, oriented so the surfaces of interest were presented to the beam. Samples were coated with a 2 nm-thin layer of Au/Pd using a Gatan ion-beam coater, and viewed using a Jeol 7401 FEG-SEM.
[0229] Chick Chorioallantoic Membrane (CAM) Assay
[0230] Fertilized chicken eggs of ‘White Leghorn’ breed (Henry Stewart and Co.) were incubated in a MultiQuip Incubator (E2) at 37° C. with 60% constant humidity.sup.38. Ethics approval was obtained by the University College London Animal Ethics Committee. A small window was made in the shell on day 3 of chick embryo development under aseptic conditions. The window was resealed with adhesive tape and eggs were returned to the incubator until day 8 of chick embryo development. On day 8, ECM gels and Matrigel grafts were placed on top of the CAM and eggs were resealed and returned to the incubator. On day 10 PBS was added to the CAM to avoid the CAM drying out. Pictures were taken on day 13 and day 15. On day 15 ECM gel and Matrigel grafts with surrounding CAM were harvested from each embryo and fixed with 4% paraformaldehyde before paraffin embedding. Serial 5 μm sections were stained with H&E. Slides were digitally scanned using the NanoZoomer (Hamamatsu Photonics K.K.).
[0231] Proteomic Sample Pre-Processing
[0232] The lyophilized ECM powder was split into 3 biological replicates, each processed independently and analyzed in triplicate by LC-MS/MS. The powder was resuspended in lysis buffer and two spike-in proteins (each at 0.5 μg/100 μg of protein powder) were added: carnitine monooxygenase oxygenase subunit (cntA, D0C9N6) from Acinetobacter baumannii, and CTP synthase (CTPsyn, Q9VUL1) from Drosophila melanogaster. Protein extraction was performed by heating at 90° C. for 10 min, and centrifuging at maximum velocity for 10 min at 4° C. ECM-derived proteins were reduced in 0.1 M dithiothreitol (DTT) at 95° C. for 5 min, dissolved in 8 M urea solution after cooling down to room temperature, alkylated with 55 mM iodoacetamide for 30 min at 25° C. in the dark. Alkylated proteins were purified using Microcon YM-10 filter unit (MRCPRT010, Millipore) for 8 times at 14000 g for 40 min.sup.39 followed by trypsin (Promega) digestion for 16 h at 37° C. pH was adjusted to 3 by addition of formic acid. Peptides were desalted by C-18 column and dried into powder and were then re-suspended in 30 μl 0.1% acetic acid for the following mass spectrometry analysis.
[0233] Proteomic LC-MS/MS Analysis
[0234] Protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed using Thermo Fusion Mass Spectrometer with Thermo Easy-nLC1000 Liquid Chromatography. 130 min of LC-MS gradients were performed by increasing organic proportion. The first level of MS was detected by Orbitrap with parameter of Resolution at 120K, Scan Rang at 300-1800 m/z, Mass Tolerance at 10 ppm. The second level of MS was isolated by Quadrupole, activated by HCD and detected by Orbitrap. The Orbitrap Resolution for the second level of MS was 30K.
[0235] Proteomic Bioinformatics Analysis
[0236] The mass spectrometry-derived data were searched against a human protein database (Uniprot Homo sapiens reference proteome, UP000005640) by MaxQuant v. 1.6.7.0.sup.40. Oxidation of methionine residues and acetyl of protein N-term were set as variable modifications. Carbamidomethyl on cysteine was set as fixed modification. Peptide-spectrum matches (PSMs) were adjusted to a 1% and then assembled further to a final protein-level false discovery rate (FDR) of 1%. Intensity-based absolute quantification (iBAQ).sup.41 was normalized according to the mean quantification of the two spiked-in proteins. Proteins with less than 2 unique peptides identified were filtered out. The remaining 1617 proteins were used in the subsequent analysis. Mean and standard error of the mean among replicates were calculated in MATLAB R2017a based on normalized iBAQ intensity values, then recalculated in percentage. Overrepresentation analyses of all and exosomal proteins was performed in DAVID 6.8.sup.42. Proteins from Gene Ontology-Cellular Component (GO-CC) categories ECM (GO-CC:0031012) and extracellular exosomes (GO-CC:0070062) were selected. A hierarchical clustering analysis of the expression of this ECM protein set in native tissues from a recent draft map of the human proteome.sup.43 (online resource available at http://www.proteomicsDB.org) was obtained using the web-based tool “Expression heatmap”.sup.44, reporting protein expressions in different tissues quantified as iBAQ at the protein level. A principal component analysis (PCA) was performed in MATLAB using log 10 iBAQ intensity values from our data and from http://www.proteomicsDB.org, both within GO-CC:0031012.
[0237] Metabolomics
[0238] 30 μL ECM powder pepsin-digested (pre-gel) were analyzed by gas chromatography—mass spectrometry (GC-MS) based metabolomics using a standard protocol.sup.45 on a Thermo Trace GC and DSQ II mass spectrometer. Chromatographic deconvolution, alignment and database matching was performed using MS-DIAL 3.90.sup.46.
[0239] Culture of Mouse Intestinal Organoids
[0240] CD1 mice and LGR5-DTR-EGFP mice.sup.30 were sacrificed by cervical dislocation and the intestine was harvested from the pylorus to the caecum. The obtained tissue was washed through once with ice-cold PBS, cleared of any mesenteric or fatty tissue and cut longitudinally. Following a further series of PBS washes, a cover slip was used to shave away the villi and the remaining tissue was cut into 2-3 mm pieces and washed vigorously. This was then incubated in 2 mM ethylenediaminetetraacetic acid (EDTA) in PBS for 30 minutes followed by vigorous shaking for 5 min in PBS. The obtained supernatant, containing the intestinal crypts, was centrifuged at 800 rpm for 5 minutes at 4° C. (Hettich zentrifugen Rotina 420). The pellet was washed once with basal media (Advanced DMEM/F12 media, supplemented with 1% of each GlutaMAX, HEPES and Penicillin/Streptomycin) and centrifuged at 1000 rpm. The pellet was re-suspended in Matrigel growth factor reduced and plated onto a 24-well plate. Primocin 1X (Thermo Fisher) and ROCK inhibitor 10 μm are added after isolation. For medium recipe look in table 2.
TABLE-US-00003 TABLE 2 Mouse small intestinal organoid medium Component Stock conc. Final conc. Advanced DMEM F-12 (Thermo 12634) — To volume HEPES (Thermo 15630080) 1M 10 mM Glutamax (Thermo 35050061) 100 X 2 mM B-27 supplement minus vitamin A 50 X 1 X (Thermo 12587010) n-acetylcysteine (Sigma A9165) 500 mM 1.25 mM Pen/Strep (Thermo 15140122) 100% 1% Wnt-3A (Peprotech 315-20) optional 50 μg/mL 100 ng/mL R-spondin 1 (Peprotech 120-38) 100 μg/mL 500 ng/mL Noggin (R&D 6057-NG) 100 μg/mL 100 ng/mL EGF (Thermo PMG8043) 500 μg/mL 50 ng/mL
[0241] Culture of Pediatric Human Intestinal and Human Gastric Organoids
[0242] Human Pediatric Samples from Small Intestine and Stomach were Collected after Consent following the guidelines of the license 08ND13 and 18DS02. Small intestinal crypt stem cells and gastric crypt stem cell were isolated from pediatric biopsies following well-established dissociation protocols.sup.47,48. Isolated crypts at first passage (p0) were cultured in Matrigel growth factor reduced droplets, or in 4 mg/mL ECM gel. For media recipes look in table 3 and 4.
TABLE-US-00004 TABLE 3 Human pediatric and fetal small intestinal organoid medium Component Stock conc. Final conc. Advanced DMEM F-12 (Thermo 12634) — To volume HEPES (Thermo 15630080) 1M 10 mM Glutamax (Thermo 35050061) 100 X 2 mM B-27 supplement minus vitamin A 50 X 1 X (Thermo 12587010) n-acetylcysteine (Sigma A9165) 500 mM 1.25 mM Pen/Strep (Thermo 15140122) 100% 1% Wnt-3A (Peprotech 315-20) 50 μg/mL 100 ng/mL R-spondin 1 (Peprotech 120-38) 100 μg/mL 500 ng/mL Noggin (R&D 6057-NG) 100 μg/mL 100 ng/mL EGF (Thermo PMG8043) 500 μg/mL 50 ng/mL Gastrin (Sigma G9020) 100 μM 10 nM GSK-3 inhibitor (CHIR 99021) 3 mM 3 μM (Tocris 4423) TGFb inhibitor (A83-01) 500 μM 500 nM (Sigma SML0788) P38 inhibitor (SB202190) (Sigma S7067) 30 mM 10 μM Prostaglandin E2 (Cambridge cay14010) 100 μM 10 nM
TABLE-US-00005 TABLE 4 Human pediatric gastric organoid medium Component Stock conc. Final conc. Advanced DMEM F-12 (Thermo 12634) — To volume HEPES (Thermo 15630080) 1M 10 mM Glutamax (Thermo 35050061) 100 X 2 mM B-27 supplement minus vitamin A 50 X 1 X (Thermo 12587010) n-acetylcysteine (Sigma A9165) 500 mM 1.25 mM Pen/Strep (Thermo 15140122) 100% 1% Wnt-3A (Peprotech 315-20) 50 μg/mL 100 ng/mL R-spondin 1 (Peprotech 120-38) 100 μg/mL 500 ng/mL Noggin (R&D 6057-NG) 100 μg/mL 100 ng/mL EGF (Thermo PMG8043) 500 μg/mL 50 ng/mL Gastrin (Sigma G9020) 100 μM 10 nM GSK-3 inhibitor (CHIR 99021) 3 mM 3 μM (Tocris 4423) TGFb inhibitor (A83-01) 500 μM 5 μM (Sigma SML0788) FGF10 (Peprotech 100-26) 100 μg/mL 200 ng/mL
[0243] Culture of Human Hepatic Organoids and Cholangiocyte Organoids
[0244] Liver organoids were cultured following the protocol previously published.sup.23,24. Hepatic organoids were split by gentle dissociation with TrypLE Express (Thermo Fisher), while ductal organoids are passaged by manual disruption. Organoids were seeded in ECM gels, Matrigel and Cultrex® 3-D Culture Matrix™ basement membrane extract (BME), both at 100% concentration, as controls. For media recipes look in table 5 and 6.
TABLE-US-00006 TABLE 5 Human ductal organoid medium Stock Final Component conc. conc. Advanced DMEM F-12 (Thermo — To volume 12634028 ) Penicillin Streptomycin (Thermo 100% 1% 15140122) L-Glutamine (Thermo 10378016) 100 X 1 X B-27 supplement (Gibco 17504-044) 50 X 1 X n-acetylcysteine (Sigma A9165) 500 mM 1.25 mM R-spondin (conditioned medium Hans 10 mL 10% Clevers Lab) EGF (Thermo PMG8043) 500 μg/mL 50 ng/mL Gastrin (Sigma G9020) 100 μM 10 nM FSK stock 10 μL 10 μM FGF10 (Peprotech 100-26) 100 μg/mL 100 ng/mL HGF (Peprotech 100-39) 20 μg/mL 25 ng/mL TGFb inhibitor (A83-01) (Sigma 5 mM 50 μM SML0788) Primocin (Thermo) 50 mg/mL 100 μg/mL Nicotinamide (Sigma 72340) 1M 10 mM
TABLE-US-00007 TABLE 6 Human hepatic organoid medium Stock Final Component conc. conc. Advanced DMEM F-12 (Thermo — To volume 12634028 ) Penicillin Streptomycin (Thermo 100% 1% 15140122) L-Glutamine (Thermo 10378016) 100 X 1 X B-27 supplement (Gibco 17504-044) 50 X 1 X R-spondin (conditioned medium Hans 10 mL 15% Clevers Lab) EGF (Thermo PMG8043) 500 μg/mL 50 ng/mL n-acetylcysteine (Sigma A9165) 500 mM 1.25 mM Gastrin (Sigma G9020) 100 μM 10 nM Nicotinamide (Sigma 72340) 1M 10 mM FGF7 (Peprotech 100-19) 50 μg/mL 100 ng/mL FGF10 (Peprotech 100-26) 100 μg/mL 100 ng/mL HGF (Peprotech 100-39) 20 μg/mL 50 ng/mL GSK-3 inhibitor (CHIR 99021) 3 mM 3 μM (Tocris 4423) TGFa (Peprotech 100-16A) 20 μg/mL 50 μg/mL Primocin (Thermo) 50 mg/mL 100 μg/mL
[0245] Culture of Human Fetal Small Intestinal Organoids and Pancreatic Ducts
[0246] Small intestines and pancreases were dissected from human fetal tissue fragments obtained immediately after termination of pregnancy from 10 to 20 PCW (post conception week), in compliance with the bioethics legislation in the UK. Fetal samples were sourced via the Joint MRC/Wellcome Trust Human Developmental Biology Resource under informed ethical consent with Research Tissue Bank ethical approval (08/H0712/34+5 and 08/H0906/21+5). For small intestines, similar isolation protocol for the mouse small intestine was adopted. For the pancreases, mesenchyme surrounding the pancreas was removed and epithelial tissue was digested in dispase II (Gibco) in Hank's balanced salt solution (HBSS; Thermo Fisher) at 37° C. for 3 min. Further dissociation was performed using collagenase P (Sigma Aldrich) with gentle pipetting. Cell clusters were rinsed once with 4 mL of advanced Dulbecco's modified Eagle's medium/nutrient mixture F12 with 1% Penicillin/Streptomycin (AdDMEM/F12; +1% P/S) and several times with DMEM/F12 +1% P/S, mixed with 30 μL of Matrigel (100% concentration), and seeded in 24-well plates. For media recipes look in table 3 and 7.
TABLE-US-00008 TABLE 7 Human fetal pancreatic organoid medium Stock Final Component conc. conc. Advanced DMEM F-12 (Thermo — To volume 12634028 ) Penicillin Streptomycin (Thermo 100% 1% 15140122) L-Glutamine (Thermo 10378016) 100 X 1 X B-27 supplement (Gibco 17504-044) 50 X 1 X N-2 supplement (Gibco 17502-048) 50 X 1 X Nicotinamide (Sigma 72340) 2M 10 mM N-Acetyl-L-cysteine (Sigma A9165) 1 mM 1 μM R-spondin 1 (Peprotech 120-38) 100 μg/mL 500 ng/mL Noggin (Peprotech 120-10C) 100 μg/mL 100 ng/mL EGF (Peprotech AF-100-15) 50 μg/mL 50 ng/mL FGF-10 (Peprotech AF-100-26) 100 μg/mL 100 ng/mL Exendin4 (Sigma E7144) 100 μM 100 nM Gastrin (Sigma G9020) 100 μM 100 nM
[0247] Passage of Organoids in ECM Gel and Matrigel
[0248] Cell were passaged every 6-8 days. To passage the organoids, ECM gel and Matrigel droplets are thoroughly disrupted by pipetting in the well and transferred to tubes in ice. Cells are washed with 10 mL of cold basal DMEM F-12+++(F-12+P/S+HEPES+Glutamax) and spin at 200 g at 4° C. Supernatant is discarded. If any ECM or Matrigel is left, wash is repeated. The pellet is resuspended in 1 mL of cold basal medium and organoids are manually disrupted by narrow (flamed) glass pipette pre-wet in BSA 1% in PBS, to avoid adhesion to the glass. Cells are washed, pelleted and supernatant is discarded. Almost-dry pellets of disaggregated organoids are included either in cold liquid Matrigel or in cold ECM equilibrated gel, aliquot in 30-40 μL droplets in Petri dishes and incubated at 37° C. for 30 minutes to form a gel. For single cell colony formation and for monolayer cell seeding, organoids pellets are treated with TrypLE Express for 5-7 min (depending on organoid size and type) at 37° C. and accurate pipetting. Disaggregated cells are washed, pelleted, and resuspended in culture medium with ROCK inhibitor. Media recipes are reported in table 2-7.
[0249] In Vivo Implantation
[0250] Animal work was ethically approved and carried out under Home Office Project Licence PPL PDD3A088A. NODSCID-gamma (NSG) mice were anaesthetized with a 2-5% isoflurane:oxygen gas mix for induction and maintenance. Pancreas organoids were embedded in ECM gel and Matrigel drops within sterile silicon O-rings (3.35×1.20). Cultures were conducted for 2-4 days before grafting subcutaneously of NSG mice. For subcutaneous transplantation, buprenorphine 0.1 mg/Kg was administered at the induction for analgesia. Under aseptic conditions a midline incision (0.5 cm) was performed on the back of the mice and the ECM gel and Matrigel drops within the O-rings were inserted in lateral pockets. Mice were sacrificed at 2.5 weeks, 4 weeks, and 8 weeks post-transplant and content of the rings fixed in 4% PFA for 1 h for histological analysis.
[0251] Quantification of Stem Cell Colonies and Organoid Diameters
[0252] To quantify the colony formation in ECM gels and Matrigel, n≥10 fields of view at 5X per replicate were acquired at the Zeiss Axio Observer Al and counted. For organoid dimension quantification, n≥30 full grown organoids were randomly quantified in different 5X fields of view per replicate. For a better approximation, 3 diameters per organoid were measured and mean diameter was considered in the final calculation.
[0253] Cell Viability Assay
[0254] Cells were passaged to ECM gel and Matrigel and seeded in quadruplicates. Enteroids were expanded for 2 days and tested for combined gel cytocompatibility. Vitality assay was performed using LIVE/DEAD™ Viability/Cytotoxicity Kit, for mammalian cells (Thermo Fisher), following supplier instructions. Briefly, organoids were washed with basal DMEM F-12 and incubated in basal medium with hoechst, calcein-AM and ethidium homodimer-1 for 45 minutes. Cells in ECM gel and Matrigel droplets were washed twice and analyzed. Hepatic organoids vitality was analyzed through Cell Titer-Glo viability assay (Promega) following manufacturer's instructions.
[0255] Immunofluorescence and Protein Quantification
[0256] ECM gel and Matrigel droplets with embedded enteroids were fixed in 2% glutaraldehyde dissolved in PBS with Ca/Mg for 1 h at room temperature, and then washed. For sections, droplets were dehydrated with sucrose 30% overnight, included in OCT and cut at the cryostat microtome in 7 μm sections. Whole mount staining was performed by blocking and permeabilizing the cells with PBS-Triton 0.5% with BSA 1%. Primary antibodies were incubated in blocking buffer for 24h at 4° C. in rotation and extensively washed. Secondary antibodies were incubated overnight at 4° C. in rotation and washed. Antibody list and dilutions are reported in table 8.
TABLE-US-00009 TABLE 8 Antibody and molecule list Antibody/conjugated molecule Dilution Ezrin (Thermo PA5-29358) 1:100 rFABP1 (R&D AF1565) 1:100 FITC-conjugated B4 isolectin (BSI-B4; Griffonia 20 μg/mL (Bandeiraea) simplicifolia) (Sigma L2895) alpha-Gal (M86) (Enzo ALX-801-090-1) 1:5 GFP (Thermo A-21311) 1:100 E-cadherin (BD 610182) 1:100 Muc-1A (Termo HM-1630-P0) 1:200 PDX-1 (Abacm AB47308) 1:200 Sox-9 (Merk AB5535) 1:500 Ki-67 (ABCAM Ab15580) 1:200 Lysozyme (Genetex GTX72913) 1:100 Lysozyme (Genetex GTX39779) 1:100 Mucin-2 (Genetex GTX100664) 1:100 Villin (Genetex GTX109940) 1:100 Olfactomedin-4 (Cell signaling 14369S) 1:50 Cytokeratin-20 (Proteintech 60183-1-lg) 1:100 Zonula occludens-1 (Invitrogen 40-2200) 1:200 Mucin-5AC (Thermo MA5-12178) 1:100 Cytokeratin-19 (Abeam AB76539) 1:100 Phalloidin 488 (Thermo A12379) 1:200 Goat anti-Rabbit 594 (Thermo A11012) 1:500 Goat anti-Rabbit 568 (Thermo A11011) 1:500 Goat anti-Rabbit 488 (Thermo A11008) 1:500 Goat anti-Mouse 488 (Thermo A11001) 1:500 Goat anti-Mouse 568 (Thermo A10037) 1:500 Donkey anti-Goat 647 (Thermo A-21447) 1:500 Anti-Guinea pig (Jackson 706-165-148) 1:500 Anti-Hamster (Abeam AB175716) 1:500 Hoechst 33342 (Thermo H1399) 10 μg/mL Calcein-AM (Thermo L3224) 3 μM Ethidium homodimer-1 (Thermo L3224) 3 μM
[0257] For human albumin protein quantification, human ductal organoids and human fetal hepatic organoids were culture for 3 days (with no medium change) and medium was collected from each well (n=4 per condition). Spent media were analyzed with Human Albumin ELISA (enzyme-linked immunosorbent assay) kit, following manufacturer's instructions.
[0258] Image Acquisition
[0259] Mouse and human organoids were imaged using a Zeiss Axio Observer Al. Stained sections were acquired on a Leica DMIL microscope and DFC420C camera, or using a Hamamatsu Photonics NanoZoomer. Immunofluorescence images of whole mount stainings and sections were acquired on a confocal microscope Zeiss LSM 710.
[0260] Bulk 3′ RNA-Sequencing
[0261] For human pediatric small intestinal organoids, RNA was isolated from cultured organoids in ECM gel and Matrigel with 20 min treatment of the droplets with Cell Recovery Solution (Corning) at 4° C. Cells were then washed in ice cold PBS to remove matrix leftovers that could interfere with RNA isolation. Organoids were centrifuged at 200 g at 4° C. and surnatant discarded. Dry pellet was lysed with RLT buffer (Qiagen). RNA was isolated with RNeasy Mini Kit (Qiagen) following manufacturer's instructions. Total RNA (100 ng) from each sample was prepared using QuantSeq 3′ mRNA-Seq Library prep kit (Lexogen GmbH) according to manufacturer's instructions. The amplified fragmented cDNA of 300 bp in size were sequenced in single-end mode using the Nova Seq 6000 (IIlumina) with a read length of 100 bp.
[0262] For human liver ductal organoids, and human fetal hepatic organoids, 5 ng of RNA/sample were used as input for the library preparation following the CEL-Seq2 technique as previously described.sup.49.
[0263] Transcriptome Bioinformatic Analyses
[0264] For human pediatric small intestinal organoids, Illumina novaSeq base call (BCL) files were converted into fastq files through bcl2fastq (version v2.20.0.422) following software guide. Sequence reads were trimmed using bbduk software (bbmap suite 37.31), following software guide, to remove adapter sequences, poly-A tails and low-quality end bases (regions with average quality below 6). Alignment was performed with STAR 2.6.0a.sup.50 on hg38 reference assembly obtained from cellRanger website (Ensembl 93), following online site guide. The expression levels of genes were determined with htseq-count 0.9.1 by using cellRanger pre-build genes annotations (Ensembl Assembly 93). All transcripts having <1 CPM in less than 4 samples and percentage of multimap alignment reads>20% simultaneously were filtered out. Differentially expressed genes (DEGs) were computed with edgeR.sup.51, using a mixed criterion based on p-value, after false discovery rate (FDR) correction by Benjamini-Hochberg method, lower than 0.05 and absolute log 2(fold change) higher than 1. A Principal Component Analysis was performed by Singular Value Decomposition (SVD) on log 2(CPM+1) data, after centering, using MATLAB R2019a (The MathWorks). Hierarchical clustering of ECM-related gene sets.sup.27 was performed with Euclidean distance and complete linkage using median-centered data, and plotted as heat maps using MATLAB. DEGs over-representation analysis of Gene Ontology (GO) categories was performed using ClueGO (version 2.5.4).sup.52.
[0265] For human liver ductal organoids, and human fetal hepatic organoids DNA library sequencing, mapping to the human reference genome and quantification of transcript abundance were performed as previously described.sup.49. Sequencing libraries were analyzed using the DESeq2 package.sup.53 in R version 3.4.0 and RStudio version 1.0.143.
[0266] Real Time PCR
[0267] cDNA was prepared using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, #4368813). Quantitative PCR detection was performed using PowerUp™ SYBR® Green Master Mix (Applied Biosystems, A25742). Assays for each sample were run in triplicate and were normalized to housekeeping gene β-actin, where data was expressed as Mean±SEM.
[0268] Stiffness Measurement
[0269] Stiffness measures were taken at the Piuma Nanoindenter (Optics 11) on Petri-dishes with 30 μL ECM gels and Matrigel droplets immersed in PBS. The probe parameters used for the measures were: tip radius 57 μm, and probe stiffness 0.44 N/m.
[0270] Co-Polymerized Hydrogels
[0271] Polyacrylamide pre-polymer is prepared by mixing acrylamide/bis-acrylamide, 40% solution 29:1 (Sigma Aldrich) with PBS −/− and photo-initiator irgacure 2959 (Ciba) solved at 35 mg/mL in methanol (Sigma Aldrich). For 1 mL of a 20% final acrylamide concentration, 100 μL of irgacure, 500 μL acr/bis-acr solution and 400 μL of PBS are mixed and kept in the dark until use. Neutralized 10 mg/mL ECM pre-gel is allowed to gelate in incubator for 30 min. The gel is then disaggregated by repetitive pipetting and thoroughly mixed with polyacrylamide pre-polymer with proportions 25-75, 50-50, 75-25. Liquid pre-gel is then polymerized between two cover glasses and a silicon ring by photoactivation at the DYM40183 BlueWave 75 UV curing spot lamp. Co-polymerized hydrogel is then extensively washed in PBS with Pen-Strep to remove any cytotoxic acrylamide monomer from the gel bulk. Prior to use for cell seeding, co-polymerized hydrogels are cut and positioned in culture wells, and pre-equilibrated with basal medium overnight.
[0272] Statistical Analysis
[0273] Statistical analyses were performed using the following software: Matlab (v. R2017a) for PCA, pie plot, bar plot, hierarchical clustering with proteomic and RNA-seq data, Microsoft Excel Professional Plus (v. 2016 MSO) to normalize and filter the proteomic data, GraphPad Prism Mac (v. 6.0h) was used with all other graphs and charts.
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