METHOD AND APPARATUS FOR MESENCHYMAL STEM CELLS ISOLATION AND PURIFICATION

Abstract

A device for isolating, retaining, separating and/or purifying mesenchymal stem cells from a heterogeneous population of cells. The device includes at least one inlet, at least one outlet, a housing, and at least one surface located in the housing, the surface having an affinity for mesenchymal stem cells, wherein the surface is in communication with the inlet and the outlet, and the at least one surface includes at least two portions facing each other, and separated by a distance ranging from 1 mm to 1 cm.

Claims

1-15. (canceled)

16. A device for isolating, retaining, separating and/or purifying mesenchymal stem cells from a heterogeneous population of cells comprising: at least one inlet; at least one outlet; a housing; at least one surface located in the housing, said surface having an affinity for mesenchymal stem cells; wherein said surface is in communication with the inlet and the outlet; and wherein the at least one surface comprises at least two portions facing each other, and separated by a distance ranging from 1 mm to 1 cm.

17. The device according to claim 16, wherein the at least one surface comprises a material selected in the group of polymeric material such as polystyrene, (PS), polyamine, polycarbonate (PC), poly-D-lysine (PDL), polycaprolactone, or inorganic material such as glass.

18. The device according to claim 16, wherein the at least one surface is smooth.

19. The device according to claim 16, wherein the at least two portions are slides or coverslips.

20. The device according to claim 16, wherein the at least two portions are facing each other in staggered rows.

21. The device according to claim 16, wherein the at least two portions are parallel to each other, or describe an angle to each other ranging from 0° to 30°.

22. The device according to claim 16, wherein the at least one surface is a planar spiral.

23. A method for isolating, retaining, separating and/or purifying mesenchymal stem cells from a heterogeneous population of cells comprising mesenchymal stem cells, comprising the following steps: pushing said heterogeneous population of cells in a device according to claim 16, at a first flow rate allowing to separate said heterogeneous population of cells into a first remaining sample comprising mesenchymal stem cells on the at least one surface and into a second resulting solution being evacuated from the device; evacuating the second resulting solution from the device; pumping a saline solution in the device at a second flow rate different from the first flow rate; and collecting the saline solution comprising isolated, retained, separated and/or purified mesenchymal stem cells; wherein the first flow rate is slower than the second flow rate; wherein the ratio between the first flow rate and the second flow rate is ranging from 2 to 50.

24. The method for isolating, retaining, separating and/or purifying mesenchymal stem cells from a heterogeneous population of cells according to claim 23, further comprising a centrifugation step before pumping the sample in the device.

25. The method for isolating, retaining, separating and/or purifying mesenchymal stem cells from a heterogeneous population of cells according to claim 23, wherein the first flow rate is ranging from 10 to 150 ml/min.

26. The method for isolating, retaining, separating and/or purifying mesenchymal stem cells from a heterogeneous population of cells according to claim 23, wherein the second flow rate is ranging from 100 to 500 ml/min.

27. A method for isolating, retaining, separating, and/or purifying mesenchymal stem cells selected from the group consisting of adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, blood-derived mesenchymal stem cells, blood umbilical cord stem cells, molar stem cells, amniotic fluid stem cells, follicular stem cells, and human embryonic stem cells (hESC) obtained without the destruction of an embryo, comprising pushing a heterogeneous population of cells in a device according to claim 16, at a first flow rate allowing to separate said heterogeneous population of cells into a first remaining sample comprising said mesenchymal stem cells on the at least one surface of the device and into a second resulting solution being evacuated from the device.

28. A system for isolation, retention, separation and/or purification of mesenchymal stem cells from at least one heterogeneous population of cells, comprising: a device according to claim 16; a first supplying reservoir fluidly connected to at least one inlet of the device and configured to comprise at least one heterogeneous population of cells comprising mesenchymal stem cells; a second supplying reservoir fluidly connected to at least one inlet of the device and configured to comprise at least one saline solution; at least one collecting reservoir fluidly connected to at least one outlet of the device; at least one pumping means fluidly connected to at least one supplying reservoir; and at least one flow controller configured to control the flow rate of a fluid pumped through the inlet of the device.

29. The system for isolation, retention, separation and/or purification of mesenchymal stem cells according to claim 28, further comprising a flowcytometer.

30. The system for isolation, retention, separation and/or purification of mesenchymal stem cells according to claim 28, further comprising a temperature controller.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0373] FIG. 1 is a device 1 according to the invention.

[0374] FIG. 2 is a device 1 according to the invention.

[0375] FIG. 3 is a device 1 according to the invention.

[0376] FIG. 4 is a device 1 according to the invention.

[0377] FIG. 5 is a device 1 according to the invention, said device 1 having a planar spiral geometry.

[0378] FIG. 6 is a histogram showing the method for isolating and purifying adipose-derived mesenchymal stem cells from a sample according to the invention.

[0379] FIG. 7 is a system 2 according to the invention.

[0380] FIG. 8 is a system 2 according to the invention, comprising a first collecting reservoir 231 and a second collecting reservoir 232.

[0381] FIG. 9 is a system 2 according to the invention, comprising a first collecting reservoir 231, a second collecting reservoir 232 and a flowcytometer 26.

[0382] FIG. 10A is a device 1 according to the invention, said device being configured to rotate.

[0383] FIG. 10B is a device 1 according to the invention, said device being configured to rotate.

[0384] FIG. 11A illustrates the flow of a cell suspension in a device 1, wherein said device does not rotate.

[0385] FIG. 11B illustrates a cell in a cell suspension in a device 1, wherein said device does not rotate.

[0386] FIG. 11C illustrates the flow of a cell suspension in a device 1, wherein said device rotates.

[0387] FIG. 11D illustrates a cell in a cell suspension in a device 1, wherein said device rotates. Said cell is much closer to the surface 14 than the cell in the non-rotating device 1.

[0388] FIG. 12 is a device 1 according to the invention, said device 1 having a parallelepiped geometry. Even though, the device is represented with a rectangular section, it may also have a square, oval, or circular section.

EXAMPLES

[0389] The present invention is further illustrated by the following examples.

Example 1

Materials and Methods

Material:

[0390] sample of adipose tissue; [0391] device 1 according to the invention.

Methods

[0392] Adipose tissue is collected by lipoaspiration. The fat tissue is added with an equal volume of saline and an equal volume of non-pyrogenic distilled water and introduced into a vortex heating chamber for 10 minutes at 39° C. The resulting suspension is centrifuged at 1200 rpm for 10 minutes. After the centrifugation round, there are a superficial layer of oil and fat tissue, an intermediate yellowish layer and a bottom pellet containing the red blood cells, fibroblasts, smooth muscle cells, white blood cells. The intermediate centrifuged layer is collected with a sterile pipette and resuspended with saline. A second centrifugation is performed at 700 rpm for 5 minutes. This results in two layers. The superficial layer containing the stem cells is collected and ready for purification. These two centrifugations allow the separation of the stem cells from the other populations.

[0393] The resulting suspension is purified using the device 1 of the invention. The suspension is flowed at a first flow rate of 100 ml/mn in the device 1. Then, a rapid flush of 300 ml/mn with saline washes out the almost adherent cells, which are collected at the end of the device 1.

Results

[0394] This results in a suspension of purified mesenchymal stem cells.

[0395] The same method was performed with a sample of bone marrow, blood umbilical cord, molar, amniotic fluid, follicular tissue (hair), or a sample from a human embryo obtained without the destruction of an embryo.

Example 2

Materials and Methods

Material:

[0396] sample of adipose tissue; [0397] device 1 according to the invention.

Methods

[0398] Adipose tissue is collected surgically during a surgical intervention of lipofilling. The fat tissue is added with an equal volume of saline and an equal volume of non pyrogenic distilled water and introduced into a vortex heating chamber for 15 minutes at 39° C. The resulting suspension is centrifuged at 1000 rpm for 14 minutes. After the centrifugation round, there are a superficial layer of oil and fat tissue, an intermediate yellowish layer and a bottom pellet containing the red blood cells, fibroblasts, smooth muscle cells, white blood cells. The intermediate centrifuged layer is collected with a sterile pipette and resuspended with saline. A second centrifugation is performed at 700 rpm for 5 minutes. This results in two layers. The superficial layer containing the stem cells is collected and ready for purification. These two centrifugations allow the separation of the stem cells from the other populations.

[0399] The resulting suspension is purified using the device 1 of the invention. The suspension is flowed at a first flow rate of 100 ml/mn in the device 1. Then, a rapid flush of 300 ml/mn with saline washes out the almost adherent cells, which are collected at the end of the device 1.

Results

[0400] This results in a suspension of purified mesenchymal stem cells.

[0401] The same method was performed with a sample of bone marrow, blood umbilical cord, molar, amniotic fluid, follicular tissue (hair), or a sample from a human embryo obtained without the destruction of an embryo.

Example 3

Materials and Methods

Material:

[0402] sample of adipose tissue; [0403] device 1 according to the invention.

Methods

[0404] Adipose tissue is collected surgically during a surgical intervention of debridement of a chronic wound. The fat tissue is added with an equal volume of saline and an equal volume of non pyrogenic distilled water and introduced into a vortex heating chamber for 10 minutes at 39° C. The resulting suspension is centrifuged at 1200 rpm for 10 minutes. After the centrifugation round, there are a superficial layer of oil and fat tissue, an intermediate yellowish layer and a bottom pellet containing the red blood cells, fibroblasts, smooth muscle cells, white blood cells. The intermediate centrifuged layer is collected with a sterile pipette and resuspended with saline. A second centrifugation is performed at 700 rpm for 5 minutes. This results in two layers. The superficial layer containing the stem cells is collected and ready for purification. These two centrifugations allow the separation of the stem cells from the other populations.

[0405] The resulting suspension is purified using the device 1 of the invention. The suspension is flowed at a first flow rate of 200 ml/mn in the device 1. Then, a rapid flush of 400 ml/mn with saline washes out the almost adherent cells, which are collected at the end of the device 1.

Results

[0406] This results in a suspension of purified mesenchymal stem cells.

[0407] The quantity of mesenchymal stem cells in the purified suspension of mesenchymal stem cells is measured using a flowcytometer 26.

[0408] The same method was performed with a sample of bone marrow, blood umbilical cord, molar, amniotic fluid, follicular tissue (hair), or a sample from a human embryo obtained without the destruction of an embryo.

REFERENCES

[0409] 1—Device for purifying mesenchymal stem cells; [0410] 11—Inlet; [0411] 12—Outlet; [0412] 13—Housing; [0413] 14—Surface; [0414] 141—Portion of surface; [0415] 142—Portion of surface; [0416] 15—rotating mechanism; [0417] 2—System; [0418] 21—First supplying reservoir; [0419] 22—Second supplying reservoir; [0420] 23—Collecting reservoir; [0421] 231—First collecting reservoir; [0422] 232—Second collecting reservoir; [0423] 24—Pumping means; [0424] 25—Flow controller; [0425] 26—Flowcytometer; [0426] 3—Flow [0427] 31—cell.