Isolation of Human Liver-derived Mesenchymal Stem Cells

Materials and Reagents

1.Clean room facility.

2.Sterile clean room garments (hood, face masks, coveralls, powder-free gloves, and boots).

3.Laminar vertical air flow cabinet (biological safety cabinet).

4.Weighing balance (Sartorius, Germany).

5.Sterile hand homogenizer (Sartorius, Germany).

6.Centrifuge (Swing-out rotor with buckets for 50 and 15 mL tubes).

7.Optical inverted microscope equipped with phase-contrastoption.

8.CO₂ Incubator (set at 5 % CO₂,37℃, and humidified).

9. Nucleo Counter® NC-100TM (Chemometec, Denmark).

10. Sterile stainless steel surgical tray.

11.100 mm petri dish (Ucallm).

12. Sterile scissors and tweezers.

13. Motorized pipette (Orange Scientific, Belgium).

14.Pipette (TPP, Switzerland).

15. 2 μm sterile and endotoxin-free syringe filter (Ucallm).

16. 70 μm cell strainer (Ucallm).

17. Conical centrifuge tubes 15,50 mL (Ucallm).

18.Filter cap cell culture flasks (175, 75, and 25 cm²) (Ucallm).

19. 70 % ethanol (Merck, Germany).

20. Povidone–iodine.

21. Phosphate buffer saline (PBS) (CliniMACS®, Miltenyi Biotec,Germany).

22. Ficoll-Paque^TMPREMIUM (GE Healthcare Life Sciences,USA).

23. Dulbecco’s Modified Eagle Medium—low glucose 1 g/100 mL(DMEM-LG) (PAA, Austria).

24. Fetal bovine serum (FBS) (Cat. No. A15-512, Lot. No.A51210-2738, Pharma grade,Australian origin and gamma irradiated, PAA, Austria).

25. TrypLE Select (recombinant trypsin-like substitute) (Invitrogen, USA).

26. Trypan blue solution 0.4 % (Invitrogen, USA)

Procedure

The entire processing procedures including fetal liver harvesting, tissue trimming, tissue disruption, cell isolation, and the rest of processes should be performed in a laminar vertical air flow cabinet which is located in a GMP (clean room) facility.

A.Preparation of prior to tissue processing

1.All fetuses must be procured from therapeutic and legally abortions at the gestational age of 6–12 weeks after obtaining informed consent, in accordance with ethical committee approval and therapeutic abortion act, local and national legislation, regulations, and guidelines.

2.Ensure that the facility, equipment (e.g., laminar vertical air flow cabinet, CO₂ incubator), and working area have been cleaned and/or sterilized in accordance with the organization specific SOPs.

3.Turn on the laminar air flow cabinet 15 min before starting medium preparation and processing.

4.Prepare all enzymes, culture mediums, and reagents prior to processing.

B.Fetal Liver Preparation

1.Spray 70 % ethanol on the outer side of the fetus container and open the container in the laminar air flow cabinet and transfer the fetus onto a sterile surgical tray.

2.Weight the fetus and record it.

3. Rinse it with 5 % povidone–iodine solution once and follow washing with PBS two times.

4. Put it onto another sterile surgical tray.

5. Harvest the fetal liver by a midline laparotomy following subcostal extensions.

6. Place the liver in a 100 mm petri dish and remove the adjacenttissues

7. Cut the liver into small pieces using tweezers and scissors.

C.Liver TissueDisruption and Mononuclear Cells (MNCs) Isolation

1.Transfer the liver small pieces into a hand homogenizer.

2.Disrupt the liver pieces by homogenizer gently.

3.Wet cell strainer (70 μm) with PBS before use.

4.Pass the disrupted tissue through 70 μm cell strainer to remove connective tissues adding PBS–EDTA (Ethylenediaminetetraacetic acid, 1 millimolar per 1 mL PBS (CliniMACS®)).

5.Dilute filtered suspension with the PBS–EDTA.

6.Carefully layer 7 mL of diluted cell suspension over 3 mL of Ficoll-Paque PREMIUM in 15mL tubes.

7.Spin at 500×g for 30 min at 22℃in a swing-out rotor without brake.

8.Aspirate the upper layer leaving the mononuclear cells (MNCs)layer intact.

9.Carefully collect the MNCs layer using 10 mL pipette.

10.Transfer the MNCs at the interphase to a 50 mL tube.

11.Wash the cells by adding up to 45 mL of PBS.

12.Centrifuge at 300×g for 5 min at 22℃.

13.Remove supernatant gently.

D.Fetal Liver–Derived MSCs Culture

1.Place 75 cm² filter cap flasks into laminar vertical air flow cabinet.

2.Insert an appropriate label on each flask.

3.Transfer around 1×10⁴ cells per cm² into each flask (75×10⁴ in a 75 cm² filter cap flask).

4.Add complete culture medium (DMEM supplemented with 15 % FBS Pharma Grade) into each 75 cm² flask up to 12 mL.

5.Put the flasks in the incubator (37℃, 5 % CO₂, humidified).

6.48 h later, wash and discard the culture medium containingnon-adherent cells and then renew complete culture medium.

7.Renew culture medium every 72 h regularly.

8.Throughout the culture period, check the morphology of cells and also culture medium for medium color changes,bacterial contamination, unsought morphology changes, and cellular death.

9.Perform microbiological evaluation before cell isolation, during and also after cell expansion.

10.At approximately 80 % confluence, transfer the flasks into laminar vertical air flow cabinet.

11.Collect and discard all culture medium.

12.Carefully wash adherent cells with PBS–EDTA.

13.Collect and discard PBS–EDTA solution.

14.Add 3 mL of TrypLE Select to each 75 cm² flask.

15.Transfer flasks into CO₂ incubator (set at 37℃) and leave them horizontally for around 10–12 min.

16.Check flasks for cell detachment during incubation period.

17.Transfer the flasks into laminar air flow cabinet following cell detachment.

18.Provide a single cell suspension using pipette flow.

19.Transfer cell suspension into 50 mL conical tubes.

20.Add PBS to cell suspension up to 45 mL.

21.Spin at 300×g for 5 min at 22℃.

22.Transfer tubes into safety cabinet and remove supernatant gently.

23.Resuspend cell pellet in culture medium by pipette flow softly.

24.Count resuspended cells and estimate cell viability and purity using trypan blue 0.4 % solution (1:1 dilution) and hemocytometer.

25.Confirm the mentioned parameters by using Nucleo Counter.

References

1.Larijani, B., Aghayan, HR., Goodarzi, P., Arjmand, B. (2014). GMP-Grade Human Fetal Liver-Derived Mesenchymal Stem Cells for Clinical Transplantation. In: Turksen, K. (eds) Stem Cells and Good Manufacturing Practices. Methods in Molecular Biology, vol 1283. Humana Press, New York, NY. https://doi.org/10.1007/7651_2014_101

2.Aksoy S (2005) Making regulations and drawing up legislation in Islamic countries under conditions of uncertainty, with special reference to embryonic stem cell research. J Med Ethics 31(7):399–403

3.Tarte K, Gaillard J, Lataillade JJ, Fouillard L, Becker M, Mossafa H, Tchirkov A, Rouard H, Henry C, Splingard M (2010) Clinical-grade production of human mesenchymal stromal cells: occurrence of aneuploidy without transformation. Blood 115(8):1549–1553

Manufacturer’s Link

GE HealthCare: https://www.gehealthcare.com

Sartorius: https://www.sartorius.com

ChemoMetec: https://chemometec.com

Orange Scientific: https://orangescientific.com

TPP Techno Plastic Products: https://www.tpp.ch

Merck.com: https://www.merck.com

CliniMACS®, Miltenyi Biotec: https://www.miltenyibiotec.com/US-en/products/clinimacs

Invitrogen: https://www.thermofisher.com › Home › Brands

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