Device for isolating stem cells from fetal tissues

Abstract

The invention relates to a device for isolating stem cells from fetal tissues, which device has an incubation chamber, at least one pump, at least one reservoir for a tissue break-down solution, at least one reservoir for a rinsing solution, optionally a control unit, optionally a means for removing contaminants, and optionally a means for expansion of the isolated stem cells. The invention further relates to a method for isolating stem cells from fetal tissue, which method comprises, among other things, the mechanical dissociation and the enzymatic digestion of the fetal tissue and optionally density gradient centrifugation for removing contaminants. The device and the method according to the invention are particularly suitable for isolating mesenchymal stem cells from fetal tissues, such as umbilical cord tissue, placenta tissue, or fetal lung tissue.

Claims

1. A device for isolating stem cells from a fetal tissue, comprising: an incubation chamber, at least one pump, at least one reservoir for a tissue-disruption solution, which is connectable to the incubation chamber, at least one reservoir for a rinse solution, which is connectable to the incubation chamber, optionally a control unit, a means for the removal of impurities in a density gradient, and optionally a means for the expansion of isolated stem cells, wherein the incubation chamber comprises a lower region having a rotating knife system for comminution of fetal tissues, wherein the rotating knife system is positioned and configured to stir the tissue-disruption solution with the fetal tissues in the lower region of the incubation chamber, wherein the accessibility of the fetal tissues for the tissue-disruption solution is ensured at all times, and wherein the device is a closed system.

2. The device as claimed in claim 1, wherein the incubation chamber is a heatable or a nonheatable tank.

3. The device as claimed in claim 1, wherein the means for the removal of impurities in a density gradient is a centrifuge.

4. The device as claimed in claim 1, wherein the incubation chamber comprises openings/ports for lines and hoses.

5. The device as claimed in claim 4, wherein the tissue-disruption solution and/or the rinse solution can be added to the incubation chamber via the openings/ports.

6. The device as claimed in claim 5, wherein the reservoir for the tissue-disruption solution and/or the reservoir for the rinse solution are connected to the incubation tank by means of hoses via the openings/ports.

7. The device as claimed in claim 1, wherein the incubation chamber comprises at least one outlet.

8. The device as claimed in claim 7, wherein a tissue-disruption solution can be transferred into other tanks via the at least one outlet.

9. The device as claimed in claim 7, wherein the at least one outlet is connected to the means for the expansion of the isolated cells by means of hoses.

10. The device as claimed in claim 1, wherein the device is the closed system, which is sterilizable.

11. The device as claimed in claim 1, wherein the at least one pump includes a peristaltic pump.

12. The device as claimed in claim 1, wherein the device further contains a means for the inline-concentration of a culture supernatant after harvesting of expanded cells.

13. The device as claimed in claim 1, wherein the fetal tissue is selected from the group consisting of umbilical cord tissue, placenta tissue, tissue from the fetal membranes, and fetal lung tissue.

14. The device as claimed in claim 1, wherein the fetal tissue is at least one entire umbilical cord.

15. The device as claimed in claim 1, wherein the stem cells are mesenchymal stem cells.

16. The device as claimed in claim 1, wherein cell-free products are obtained.

17. The device as claimed in claim 1, wherein the at least one reservoir for the tissue-disruption solution contains the tissue-disruption solution, wherein the tissue-disruption solution contains a mixture of enzymes hyaluronidase and neutral protease and/or collagenase and/or DNAse; or a mixture of the enzymes DNAse and hyaluronidase and/or neutral protease and/or collagenase.

18. A system comprising: the device of claim 1; and the tissue-disruption solution, which contains a mixture of enzymes hyaluronidase and neutral protease and/or collagenase and/or DNAse; or a mixture of the enzymes DNAse and hyaluronidase and/or neutral protease and/or collagenase.

Description

(1) The invention will be elucidated below on the basis of three figures and one exemplary embodiment.

(2) FIG. 1 shows the device 10 according to the invention as a closed system. The device comprises an incubation chamber 11 with lid 28, a reservoir for the tissue-disruption solution 12 and a reservoir for the rinse solution 13. The reservoirs for the tissue-disruption solution 12 and the rinse solution 13 are both connected to the incubation chamber 11 via hoses 20 and the ports 16 and 17. The tissue-disruption solution can be conveyed from the reservoir 12 into the incubation chamber 11 by means of the peristaltic pump 14. The rinse solution can be filled into the incubation chamber 11 from the reservoir 13 by means of the peristaltic pump 15. The incubation chamber 11 contains a rotating cutting knife 21 for the mechanical dissociation of the fetal tissue. The temperature in the incubation chamber 11 can be regulated by means of an electric heating rod 24 and a temperature sensor 23. Alternatively, the temperature in the incubation chamber can also be regulated by means of a Peltier element provided with a sterilizable or disposable shell. The pH of the tissue-disruption solution in the incubation chamber 11 can be monitored by means of the optional pH probe 22. The incubation chamber 11 additionally has an outlet 18 which is connected via hoses 20 to a HYPERStack® system from Corning for the subsequent adherent cell expansion. After performance of mechanical dissociation and enzymatic digestion, the tissue-disruption suspension can be transferred from the incubation chamber 11 into the HYPERStack® System 25 by means of the peristaltic pump 19. The device 10 can be controlled using a smartphone or tablet PC 29 as control unit. Said control unit can, for example, be connected wirelessly to the device 10.

(3) FIG. 2 shows a device according to the invention that is realized as a closed system, as in FIG. 1. In contrast to the embodiment in FIG. 1, the device according to FIG. 2 contains a density-gradient centrifuge 26 instead of the HYPERStack® system 25.

(4) Alternatively, the device according to the invention can also contain a density-gradient centrifuge 26 and a HYPERStack® system 25.

(5) FIG. 3 shows one embodiment of the device according to the invention as a closed system, as shown in FIG. 1. In contrast to FIG. 1, the temperature in the incubation chamber 11 is adjusted by means of a heating plate 27 instead of an electric heating rod 24. Alternatively, the temperature in the incubation chamber can also be regulated by means of a Peltier element provided with a sterilizable or disposable shell.

(6) The device 10 can be controlled using a PC 30 as control unit, with PC 30 and device 10 being, for example, cable-connected via the cable 31.

EXEMPLARY EMBODIMENT: ISOLATION OF A BATCH OF PRIMARY MSCS FROM AN UMBILICAL CORD

(7) A batch of MSCs was obtained from an umbilical cord using the device according to the invention and the method according to the invention. To this end, the umbilical cord was first stored under controlled conditions until the transport to the device 10 according to the invention. The storage was done at a temperature of 4° C. and in a specific medium composed of a citrate-phosphate-dextrose-adenine solution (CPDA1) and PBS (CDPA1/PBS).

(8) The transport to the device 10 according to the invention was likewise done under controlled conditions at a temperature of 4° C. in the medium CPDA1/PBS.

(9) The isolation of the MSCs from the umbilical cord was then carried out in the device 10 according to the invention. To this end, the entire umbilical cord was placed into the incubation tank 11, which was closed with the lid 28. Before the performance of the MSC isolation, the entire device 10 was sterilized. The mechanical disruption of the umbilical cord was done automatically by means of the rotating knife system 21 after closure of the lid 28 in the incubation chamber 11.

(10) This was followed by the enzymatic dissociation using an AoF enzyme mixture which contained recombinant collagenase, hyaluronidase and DNAse of eukaryotic origin. The DNAse was Pulmozyme (Roche Diagnostics). The collagenase used was collagenase NB IV/VI (SERVA Electrophoresis). The tissue-disruption solution (50 ml) additionally contained Dulbecco's modified Eagle medium (DMEM). Also present in the tissue-disruption solution were glucose and HEPES. The tissue-disruption solution was introduced into the incubation chamber from the reservoir 12 via the inlet 16 by means of peristaltic pump 14.

(11) The enzymatic dissociation, which was carried out for 3 h, was followed by a density-gradient separation in a CEPA centrifuge (Carl Padberg, Zentrifugenbau GmbH) 26. To this end, the incubation chamber 11 was first rinsed by introduction of 50 ml of rinse solution (Dulbecco's phosphate buffered saline (PBS)) from the reservoir 13 by means of the peristaltic pump 15. Via the outlet 18 and by means of the peristaltic pump 19, the tissue-disruption solution was then transferred into the density-gradient centrifuge 26 via the hoses 20. The density-gradient centrifugation was carried out using Ficol® tubes. Performance of the density-gradient separation was followed by the transfer and seeding of the primary culture into a closed cell-culture system from HYPERStack® (Corning) 25. The yield before the expansion of the target cells was approx. 1×10.sup.6 cells per umbilical cord. The subsequent cell expansion, necessary media change and the cell harvesting was done with AoF reagents in the closed system of the device 10. Altogether only two passages were required in order to reach a target cell count of 1×10.sup.9 cells. The cell harvesting was followed by the cryopreservation. A sterility test, a FACS analysis and a potency assay were carried out for the quality control.

(12) The target cells of the thus worked-up MSC batch bear the surface markers CD73, CD90 and CD105, but do not bear the surface markers CD14, CD34 and CD45. With the purification of a batch of MSCs from an umbilical cord that was carried out by means of the device 10 according to the invention, it was possible to achieve a purity of greater than 95% of the target cells bearing the surface markers CD73, CD90 and CD105. Less than 2% of the purified cells bore the unwanted markers CD14, CD34 and CD45.

(13) The table below illustrates the process for producing a batch of MSCs from umbilical cord tissue using the device according to the invention.

(14) TABLE-US-00001 Invention Prior art Obtaining the Controlled storage until transport Uncontrolled storage/not starting material (specif. temperature and specif. specified (umbilical cord) medium) Controlled transport for work-up Uncontrolled transport/not (specif. temperature and specif. specified medium) Isolation of the Automatic, mechanical dissociation Manual processing by e.g. MSC from the Enzymatic dissociation with scratching of the surface or umbilical cord specif. AoF enzyme cocktail manual dissociation Density-gradient separation with — specif. separation medium Seeding of primary culture in Plating out of the tissue pieces closed cell-culture system in Petri dishes (open system) Expansion Cell expansion, medium change and Cell expansion, medium change and cell harvesting with AoF reagents cell harvesting, in some cases in completely closed system with calf serum and trypsin, in open system Low number of passages necessary Distinctly more passages (<P3) for reaching target cell necessary (>P5) for reaching count of 1 × 10{circumflex over ( )}9 cells target cell count of 1 × 10{circumflex over ( )}9 cells Cryopreservation Cryopreservation Cryopreservation Quality control Sterility test Sterility test FACS analysis with extended panel FACS analysis with standard panel Potency assay — Yield Approx. 1 × 10{circumflex over ( )}6 cells/umbilical Approx. 1 × 10{circumflex over ( )}5 cells/umbilical cord cord Purity >95% >80% (CD73+, CD90+, CD105+) Purity <2% <10% (CD14−, CD34−, CD45−)

LIST OF REFERENCE SIGNS

(15) 10 Device for isolating stem cells from fetal tissues 11 Incubation chamber 12 Reservoir for tissue-disruption solution 13 Reservoir for rinse solution 14 Peristaltic pump for pumping the tissue-disruption solution 15 Peristaltic pump for pumping the rinse solution 16 Port for the tissue-disruption solution 17 Port for the rinse solution 18 Outlet 19 Peristaltic pump for pumping the tissue-disruption suspension 20 Hoses 21 Rotating knife system 22 pH probe 23 Temperature sensor 24 Electric heating rod or Peltier element 25 HYPERStack® system 26 Density-gradient centrifuge 27 Electric heating plate 28 Lid 29 Smartphone, tablet PC 30 PC 31 Cable

REFERENCES

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