Methods in Molecular Biology (2015) 1212: 65–72 DOI 10.1007/7651_2014_174 © Springer Science+Business Media New York 2014 Published online: 04 January 2015
Porous Membrane Culture Method for Expansion of Human Pluripotent Stem Cells Jin-Su Kim, Seung-Taeh Hwang, and Soo-Hong Lee Abstract For the clinical application of human pluripotent stem cells (hPSCs), it is critical to develop novel culture techniques that completely exclude the use of animal feeder cells and enzyme treatments used in conventional hPSC culture systems. Here, we describe a novel culture method using a porous membrane that allows to maintain stable attachment and expansion of hPSCs, obviates the need for enzyme treatment, and also reduces feeder layer contamination. Keywords: hPSCs, Self-renewal, Porous membrane, Co-culture, Expansion, Proliferative human feeder cell
1
Introduction Human pluripotent stem cells (hPSCs) including human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) have pluripotent differentiation capacity and indefinite self-renewal potential. Therefore hPSCs have attracted great attention for their potential applications in regenerative medicine such as cell replacement therapies, tissue engineering, and drug discovery (1–3). Since Thomson’s and Yamanaka’s groups first succeeded in the hESCs and hiPSCs using mouse feeder cells in the presence of mitomycin C (4, 5), a variety of other culture methods have been suggested to maintain the self-renewal capacity of hPSCs. However, many researchers still follow the conventional feeder cell-based culture methods because feeder cells provide mechanical support and secrete soluble factors necessary for hPSC expansion and stemness (6–8). To avoid contamination with animal feeder cells, several studies have assessed human feeder cells such as human dermal fibroblasts (HDFs), bone marrow-derived mesenchymal stem cells (BMMSCs), and placental cells (9–11). However, conventional cultures using human feeder cells still require suppression of feeder cell growth using mitomycin C and enzyme treatment during hPSC transfer for expansion. Recently, feeder-free culture methods have been established by using specific
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medium and extracellular matrix components (12, 13), but these methods are often difficult for long-term hPSC cultures; furthermore, they may potentially give rise to genetic aberrations in hPSCs by increasing chromosomal instability and susceptibility to mitochondrial diseases (14–17). On the other hand, suspension culture methods have the benefit of enabling mass production of hPSCs (18, 19). Nonetheless, such suspension cultures have not yet been established. For future clinical applications of hPSCs, formulaic methods for hPSC long-term cultures are required that do not use animal feeder cells and enzyme treatments. Here, we describe a novel culture method for hPSC expansion that uses a porous membrane (PM) insert (1 μM) and proliferative human adipose-derived stem cells (hASCs) as a feeder layer (20–22). Proliferative hASCs were seeded onto the bottom surface of an inverted PM insert and hPSCs were subsequently cultured on the top surface of the PM insert. Because hPSC and hASC feeder cells are physically separated from each other, hPSCs can be easily mechanically transferred during subculture without enzyme treatment. hPSCs cultured on PM exhibit strong expression of stemness markers and normal karyotype and maintain pluripotency, as evidenced by their ability to form teratomas with three germ layers in vivo. This culture method would be very useful for clinical-grade hPSCs with self-renewal, because it excludes enzyme treatment and suppression of feeder cells.
2
Materials Prepare all reagents at room temperature and store in 4 C refrigerator (unless indicated otherwise). Industriously accomplish all waste disposal regulations when disposing waste materials.
2.1 hASC Isolation and Expansion
1. Human adipose tissue: Under GMP conditions in the CHA Stem Cell Institute with approval from the Institutional Review Board of CHA University Hospital Ethics Committee (IRB No. PBC09-099). 2. 10- and 25-ml sterile serological pipettes (SPL, cat. no. 1010, 91025). 3. 15- and 50-ml sterile polypropylene centrifuge tubes (SPL, cat. no. 50015, 50050). 4. Antibiotics: Penicillin/streptomycin (P/S; Hyclone, cat. no. SV30010.) and antibiotics-antimycotics (A/A; Hyclone, cat. no. SV30079.01). 5. Filter 0.22-μm low protein-binding sterilization unit (Corning, cat. no. 430769). 6. Type II collagenase (Sigma, cat. no. C6885-1G).
Porous Membrane Culture Method for Expansion of Human Pluripotent Stem Cells
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7. Dulbecco’s Phosphate-Buffered Saline (DPBS; Hyclone, cat. no. SH30028.02). 8. Hank’s Balanced Salt Solution (HBSS; Hyclone, cat. no. SH30588.01). 9. Fetal bovine serum (FBS; Hyclone, SH30070.01) (see Note 1). 10. 100-mm cell culture dish (TPP, cat. no. 93100). 11. EDTA (0.05/EDTA (0.05 % (wt/vol), Hyclone, cat. no. SH30042.01). 12. Freezing container (Nalgene, cat. no. 500-0001). 13. 1.5-ml cryogenic vials (Nunc, cat. no. 3668632). 14. DMSO (Sigma, cat. no. D2650). 15. Micro forceps (Jeung-do bio, cat. no. JD-S-27). 16. Dulbecco’s modified eagle medium (DMEM)-high glucose (Hyclone, cat. no. SH30243.01). 17. Washing solution: Add 2 % (v/v) A/A in HBSS. 18. Type II collagenase solution: Add 0.1 % (wt/vol) collagenase in DPBS containing 1 % BSA and 2 mM calcium chloride. 19. hASC culture medium: Add 10 % FBS and 1 % P/S in DMEMhigh glucose. 20. Cell stock medium: Mix 50 % FBS, 40 % DMEM-H, and 10 % DMSO (see Note 2). 2.2 Preparation of Feeder Cell on PM Insert
1. Glass beaker (50 ml, Beckman, cat. no. 4110003). 2. PM insert (BD Falcon, cat. no. 353102). 3. Microforceps (Jeung-do bio, cat. no. JD-S-27). 4. Teflon ring (customized). 5. 6-well plate (Falcon, cat. no. 353046). 6. Trypsin/EDTA SH30042.01).
(0.05 %
(wt/vol), Hyclone,
cat.
no.
7. Hemocytometer (Marienfeld, cat. no. 100612). 8. Washing solution: Add 2 % (v/v) A/A in HBSS. 2.3 hPSCs Expansion on PM Insert
1. DMEM/F12 medium (Life Technologies, cat. no. 11320033). 2. Knockout serum replacement (KSR; Life Technologies, cat. no. 10828-028). 3. L-Glutamine (Life Technologies, cat. no. 35050-061). 4. ß-Mercaptoethanol (Life Technologies, cat. no. 21985-023). 5. DPBS (Hyclone, cat. no. SH30028.02). 6. Basic fibroblast growth factor (bFGF; Life Technologies, 13256-029).
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7. Bovine serum albumin (BSA; Sigma, A2153). 8. Nonessential amino acids (Life Technologies, cat. no. 11140050). 9. Gelatin powder (Sigma, cat. no. G9391). 10. 0.1 % gelatin solution: Add 0.1 % (wt/vol) gelatin powder in ultrapure water. Boil gelatin mixture using autoclave and filter gelatin solution. 11. bFGF stock solution: Aliquot and store at 20 C for up to 6 months. Add 10 μg bFGF to 1 ml 0.1 % BSA in PBS with CaCl2 and MgCl2. 12. hPSC culture medium: Filter sterilize and store at 4 C. Medium will last for up to 14 days. Add 20 % KSR, 1 % P/S, 1 % NEAA, 0.1 mM ß-mercaptoethanol, and 4 ng/ml bFGF in DMEM/F12.
3
Methods All procedures were performed on a clean bench unless otherwise specified.
3.1 hASC Isolation and Expansion
1. Place a maximum of 25 ml of adipose tissue into a sterile 50-ml centrifuge tube. 2. To wash off the lipoaspirate, add an equal volume of HBSS with 1 % antibiotics, and then shake vigorously for 10 min. 3. Put the tube on a clean bench and carefully remove the HBSS solution using a pipette. 4. Repeat the above washing procedure three times (see Note 3). 5. Transfer washed adipose tissue to a new sterile tube and digest with 0.1 % collagenase for 45 min at 37 C by using an intermittent shaking incubator (see Note 4). 6. After the digestion, remove the floating mature adipocyte layer using a pipette and discard it. 7. Filter the remaining solution through 40-μm cell strainers. 8. Centrifuge the cells at 300 g for 5 min. 9. Aspirate the supernatants, leaving 5 ml of solution (see Note 5). 10. Resuspend each cell pellet in 10 ml of hASC medium. 11. Centrifuge the cells at 300 g for 5 min. 12. Aspirate the supernatants, leaving 5 ml of hASC medium. 13. Repeat the above washing procedure twice. 14. Resuspend isolated cells in 5 ml of hASC medium and then seed the cell suspension onto a 100-mm cell culture dish. 15. Incubate in a humidified air incubator at 37 C with 5 % CO2.
Porous Membrane Culture Method for Expansion of Human Pluripotent Stem Cells
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16. The next day, change the medium to wash away any nonadherent cells. 17. Observe the cells daily using an inverted phase-contrast microscope. 18. Feed cells every 2 days by replacing the hASC medium. 19. Subculture cells using trypsin when hASCs reach 80–90 % confluence. 20. Split hASCs weekly at a 1:3 ratio. 3.2 Preparation of Feeder Cell on PM Insert (Scheme 1)
1. Pour 50 ml of hASC medium into a glass beaker. 2. Put the PM insert with an inverted status into a glass beaker and then put into a Teflon ring on the PM insert (see Note 6). 3. Remove extra hASC medium at the bottom of the PM insert. 4. After the preparation of PM insert, remove the hASC medium from a feeder plate. 5. Wash the plate twice with DPBS solution. 6. Dissociate the trypsinization.
hASCs
using
standard
methods
of
7. Incubate at 37 C incubator for 3 min. 8. Add 10 ml of hASC medium and pipette vigorously to disperse the hASC aggregates. 9. Count hASCs using a hemocytometer (see Note 7).
Scheme 1 Scheme for the preparation of feeder cells on PMs insert
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10. Centrifuge the counted cells at 300 g for 5 min. 11. Resuspend the cell pellet in 2 ml of hASC medium. 12. Gently pipette the cells few times up and down in the tube. 13. Seed hASCs onto the bottom of inverted PM at a density of 2 103 cells/cm2. 14. Incubate in a humidified air incubator at 37 C with 5 % CO2 for 24 h. 15. Remove the Teflon ring from the insert and observe hASCs at the bottom surface of the PM insert under a microscope. 16. Wash “the PM insert with hASCs feeder cells” using DPBS solution and move it to a 6-well plate. 3.3 hPSC Expansion on PM Insert (Scheme 2)
1. Prepare “the PM insert with hASCs feeder cells” as described above. 2. Add 5 ml of hPSC medium into each well containing the PM insert. 3. Transfer 60 hPSC clumps onto the PM insert using the mechanical transfer method (23) (see Note 8). 4. Label the 6-well plate with the hPSC line name, passage number, date, and your initials. 5. Place the plate into a humidified air incubator at 37 C with 5 % CO2 and then gently shake the plate (see Note 9). 6. After a 48-h incubation, observe hPSCs under a microscope. 7. Discard the spent medium with undetached cells and debris. 8. Gently add fresh hPSC culture medium to the PM insert. 9. Move the 6-well plate back into the humidified air incubator at 37 C with 5 % CO2.
Scheme 2 Scheme for hPSC maintain using proliferative feeder-based PMs insert culture systems
Porous Membrane Culture Method for Expansion of Human Pluripotent Stem Cells
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10. Refresh hPSC culture medium daily until cells require transfer. 11. After 6 total days of hPSCs culture, mechanically isolate fully grown hPSCs. 12. Passage hPSCs onto a new PM insert with fresh hASC feeder cells at a 1:5 ratio.
4
Notes 1. We advise using the same lot of FBS for the entire series of experiments. 2. We recommend preparing the cell stock solution on the same day and keeping it on ice. 3. Wash off the lipoaspirate extensively to remove most leukocytes and erythrocytes. If the medium from the final wash is still red, wash again by repeating steps 2–3. 4. We dissolve the required amount of collagenase in 50 ml of DPBS containing 1 % BSA and 2 mM CaCl2 and then filter sterilize it into the remaining working volume. Adipose tissue is shaken by hand every 10 min. 5. Be careful while handling cell pellets, as they are gelatinous and do not adhere readily to the centrifuge tubes. 6. Check that there is no medium leakage. This is important, because cells should attach to the bottom of PM with no significant loss. 7. For proliferative hASC feeder-based PM culture systems, we recommend a standard hASC plating density of 1–3 103 cells/cm2. A much higher initial feeder density may reduce the hPSC colony attachment rate. 8. This can be done by using a modified pipette, PIPETMAN tip, needle, or SweMed™ instrument. 9. We recommend to gently shake the plate side to side and back and forth to evenly distribute the cells. Avoid circular motions to prevent cell pooling in the center of the well.
Acknowledgment This work was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, and Future Planning (NRF-2013R-1A2A-1A09013980 and NRF-2012M3A9B4028569).
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References 1. Hentze H, Graichen R, Colman A et al (2007) Cell therapy and the safety of embryonic stem cell-derived grafts. Trends Biotechnol 25:24–32 2. Xu X, Browning VL, Odorico JS et al (2011) Activin, BMP and FGF pathways cooperate to promote endoderm and pancreatic lineage cell differentiation from human embryonic stem cells. Mech Dev 128:412–427 3. Liang YJ, Yang BC, Chen JM et al (2011) Changes in glycosphingolipid composition during differentiation of human embryonic stem cells to ectodermal or endodermal lineages. Stem Cells 29:1995–2004 4. Thomson JA, Itskovitz-Eldor J, Shapiro SS et al (1998) Embryonic stem cell lines derived from human blastocysts. Science 282 (5391):1145e7 5. Kazutoshi T, Koji T, Mari O et al (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131 (5):861–872 6. Archambeault DR, Yao HH (2010) Activin A, a product of fetal Leydig cells, is a unique paracrine regulator of Sertoli cell proliferation and fetal testis cord expansion. Proc Natl Acad Sci U S A 107:10526–10531 7. Wang L, Schulz TC, Sherrer ES et al (2007) Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling. Blood 110:4111–4119 8. Ma W, Tavakoli T, Derby E et al (2008) Cell extracellular matrix interactions regulate neural differentiation of human embryonic stem cells. BMC Dev Biol 8:90 9. Richards M, Fong CY, Chan WK et al (2002) Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat Biotechnol 20 (9):933e6 10. Cheng L, Hammond H, Ye Z et al (2003) Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture. Stem Cells 21(2):131e42 11. Genbacev O, Krtolica A, Zdravkovic T et al (2005) Serum-free derivation of human embryonic stem cell lines on human placental fibroblast feeders. Fertil Steril 83(5):151729 12. Xu C, Inokuma MS, Denham J et al (2001) Feeder-free growth of undifferentiated human
embryonic stem cells. Nat Biotechnol 19 (10):971e4 13. Hiquchi A, Ling QG, Lo YA et al (2011) Biomaterials for the feeder-free culture of human embryonic stem cells and induced pluripotent stem cells. Chem Rev 111(5):3021–3035 14. Catalina P, Montes R, Ligero G et al (2008) Human ESCs predisposition to karyotypic instability: is a matter of culture adaptation or differential vulnerability among hESC lines due to inherent properties? Mol Cancer 7:76 15. Ramos-Mejia V, Bueno C, Roldan M et al (2011) The adaptation of human embryonic stem cells to different feeder-free culture conditions is accompanied by a mitochondrial response. Stem Cells Dev 21 (7):1145–1155 16. Baker DE, Harrison NJ, Maltby E et al (2007) Adaptation to culture of human embryonic stem cells and oncogenesis in vivo. Nat Biotechnol 25(2):207e15 17. Maitra A, Arking DE, Shivapurkar N et al (2005) Genomic alterations in cultured human embryonic stem cells. Nat Genet 37 (10):1099e103 18. Steiner D, Khaner H, Cohen M et al (2010) Derivation, propagation and controlled differentiation of human embryonic stem cells in suspension. Nat Biotechnol 28:361–364 19. Amit M, Laevsky I, Miropolsky Y et al (2011) Dynamic suspension culture for scalable expansion of undifferentiated human pluripotent stem cells. Nat Protocol 6:572–579 20. Hwang ST, Lee SJ, Kang SW et al (2009) Human embryonic stem cell culture technique on mitomycin C-free feeder cells using porous membrane. Tissue Eng Regener Med 6:1375–1380 21. Kim S, Ahn SE, Lee JH et al (2007) A novel culture technique for human embryonic stem cells using porous membranes. Stem Cells 25 (10):2601–2609 22. Hwang ST, Kang SW, Lee SJ et al (2010) The expansion of human ES and iPS cells on porous membranes and proliferating human adipose-derived feeder cells. Biomaterials 31:8012–8021 23. Oh SK, Kim HS, Park YB et al (2005) Methods for expansion of human embryonic stem cells. Stem Cells 23(5):605–609
Porous Membrane Culture Method for Expansion of Human Pluripotent Stem Cells Jin-Su Kim, Seung-Taeh Hwang, and Soo-Hong Lee Abstract For the clinical application of human pluripotent stem cells (hPSCs), it is critical to develop novel culture techniques that completely exclude the use of animal feeder cells and enzyme treatments used in conventional hPSC culture systems. Here, we describe a novel culture method using a porous membrane that allows to maintain stable attachment and expansion of hPSCs, obviates the need for enzyme treatment, and also reduces feeder layer contamination. Keywords: hPSCs, Self-renewal, Porous membrane, Co-culture, Expansion, Proliferative human feeder cell
1
Introduction Human pluripotent stem cells (hPSCs) including human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) have pluripotent differentiation capacity and indefinite self-renewal potential. Therefore hPSCs have attracted great attention for their potential applications in regenerative medicine such as cell replacement therapies, tissue engineering, and drug discovery (1–3). Since Thomson’s and Yamanaka’s groups first succeeded in the hESCs and hiPSCs using mouse feeder cells in the presence of mitomycin C (4, 5), a variety of other culture methods have been suggested to maintain the self-renewal capacity of hPSCs. However, many researchers still follow the conventional feeder cell-based culture methods because feeder cells provide mechanical support and secrete soluble factors necessary for hPSC expansion and stemness (6–8). To avoid contamination with animal feeder cells, several studies have assessed human feeder cells such as human dermal fibroblasts (HDFs), bone marrow-derived mesenchymal stem cells (BMMSCs), and placental cells (9–11). However, conventional cultures using human feeder cells still require suppression of feeder cell growth using mitomycin C and enzyme treatment during hPSC transfer for expansion. Recently, feeder-free culture methods have been established by using specific
65
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medium and extracellular matrix components (12, 13), but these methods are often difficult for long-term hPSC cultures; furthermore, they may potentially give rise to genetic aberrations in hPSCs by increasing chromosomal instability and susceptibility to mitochondrial diseases (14–17). On the other hand, suspension culture methods have the benefit of enabling mass production of hPSCs (18, 19). Nonetheless, such suspension cultures have not yet been established. For future clinical applications of hPSCs, formulaic methods for hPSC long-term cultures are required that do not use animal feeder cells and enzyme treatments. Here, we describe a novel culture method for hPSC expansion that uses a porous membrane (PM) insert (1 μM) and proliferative human adipose-derived stem cells (hASCs) as a feeder layer (20–22). Proliferative hASCs were seeded onto the bottom surface of an inverted PM insert and hPSCs were subsequently cultured on the top surface of the PM insert. Because hPSC and hASC feeder cells are physically separated from each other, hPSCs can be easily mechanically transferred during subculture without enzyme treatment. hPSCs cultured on PM exhibit strong expression of stemness markers and normal karyotype and maintain pluripotency, as evidenced by their ability to form teratomas with three germ layers in vivo. This culture method would be very useful for clinical-grade hPSCs with self-renewal, because it excludes enzyme treatment and suppression of feeder cells.
2
Materials Prepare all reagents at room temperature and store in 4 C refrigerator (unless indicated otherwise). Industriously accomplish all waste disposal regulations when disposing waste materials.
2.1 hASC Isolation and Expansion
1. Human adipose tissue: Under GMP conditions in the CHA Stem Cell Institute with approval from the Institutional Review Board of CHA University Hospital Ethics Committee (IRB No. PBC09-099). 2. 10- and 25-ml sterile serological pipettes (SPL, cat. no. 1010, 91025). 3. 15- and 50-ml sterile polypropylene centrifuge tubes (SPL, cat. no. 50015, 50050). 4. Antibiotics: Penicillin/streptomycin (P/S; Hyclone, cat. no. SV30010.) and antibiotics-antimycotics (A/A; Hyclone, cat. no. SV30079.01). 5. Filter 0.22-μm low protein-binding sterilization unit (Corning, cat. no. 430769). 6. Type II collagenase (Sigma, cat. no. C6885-1G).
Porous Membrane Culture Method for Expansion of Human Pluripotent Stem Cells
67
7. Dulbecco’s Phosphate-Buffered Saline (DPBS; Hyclone, cat. no. SH30028.02). 8. Hank’s Balanced Salt Solution (HBSS; Hyclone, cat. no. SH30588.01). 9. Fetal bovine serum (FBS; Hyclone, SH30070.01) (see Note 1). 10. 100-mm cell culture dish (TPP, cat. no. 93100). 11. EDTA (0.05/EDTA (0.05 % (wt/vol), Hyclone, cat. no. SH30042.01). 12. Freezing container (Nalgene, cat. no. 500-0001). 13. 1.5-ml cryogenic vials (Nunc, cat. no. 3668632). 14. DMSO (Sigma, cat. no. D2650). 15. Micro forceps (Jeung-do bio, cat. no. JD-S-27). 16. Dulbecco’s modified eagle medium (DMEM)-high glucose (Hyclone, cat. no. SH30243.01). 17. Washing solution: Add 2 % (v/v) A/A in HBSS. 18. Type II collagenase solution: Add 0.1 % (wt/vol) collagenase in DPBS containing 1 % BSA and 2 mM calcium chloride. 19. hASC culture medium: Add 10 % FBS and 1 % P/S in DMEMhigh glucose. 20. Cell stock medium: Mix 50 % FBS, 40 % DMEM-H, and 10 % DMSO (see Note 2). 2.2 Preparation of Feeder Cell on PM Insert
1. Glass beaker (50 ml, Beckman, cat. no. 4110003). 2. PM insert (BD Falcon, cat. no. 353102). 3. Microforceps (Jeung-do bio, cat. no. JD-S-27). 4. Teflon ring (customized). 5. 6-well plate (Falcon, cat. no. 353046). 6. Trypsin/EDTA SH30042.01).
(0.05 %
(wt/vol), Hyclone,
cat.
no.
7. Hemocytometer (Marienfeld, cat. no. 100612). 8. Washing solution: Add 2 % (v/v) A/A in HBSS. 2.3 hPSCs Expansion on PM Insert
1. DMEM/F12 medium (Life Technologies, cat. no. 11320033). 2. Knockout serum replacement (KSR; Life Technologies, cat. no. 10828-028). 3. L-Glutamine (Life Technologies, cat. no. 35050-061). 4. ß-Mercaptoethanol (Life Technologies, cat. no. 21985-023). 5. DPBS (Hyclone, cat. no. SH30028.02). 6. Basic fibroblast growth factor (bFGF; Life Technologies, 13256-029).
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7. Bovine serum albumin (BSA; Sigma, A2153). 8. Nonessential amino acids (Life Technologies, cat. no. 11140050). 9. Gelatin powder (Sigma, cat. no. G9391). 10. 0.1 % gelatin solution: Add 0.1 % (wt/vol) gelatin powder in ultrapure water. Boil gelatin mixture using autoclave and filter gelatin solution. 11. bFGF stock solution: Aliquot and store at 20 C for up to 6 months. Add 10 μg bFGF to 1 ml 0.1 % BSA in PBS with CaCl2 and MgCl2. 12. hPSC culture medium: Filter sterilize and store at 4 C. Medium will last for up to 14 days. Add 20 % KSR, 1 % P/S, 1 % NEAA, 0.1 mM ß-mercaptoethanol, and 4 ng/ml bFGF in DMEM/F12.
3
Methods All procedures were performed on a clean bench unless otherwise specified.
3.1 hASC Isolation and Expansion
1. Place a maximum of 25 ml of adipose tissue into a sterile 50-ml centrifuge tube. 2. To wash off the lipoaspirate, add an equal volume of HBSS with 1 % antibiotics, and then shake vigorously for 10 min. 3. Put the tube on a clean bench and carefully remove the HBSS solution using a pipette. 4. Repeat the above washing procedure three times (see Note 3). 5. Transfer washed adipose tissue to a new sterile tube and digest with 0.1 % collagenase for 45 min at 37 C by using an intermittent shaking incubator (see Note 4). 6. After the digestion, remove the floating mature adipocyte layer using a pipette and discard it. 7. Filter the remaining solution through 40-μm cell strainers. 8. Centrifuge the cells at 300 g for 5 min. 9. Aspirate the supernatants, leaving 5 ml of solution (see Note 5). 10. Resuspend each cell pellet in 10 ml of hASC medium. 11. Centrifuge the cells at 300 g for 5 min. 12. Aspirate the supernatants, leaving 5 ml of hASC medium. 13. Repeat the above washing procedure twice. 14. Resuspend isolated cells in 5 ml of hASC medium and then seed the cell suspension onto a 100-mm cell culture dish. 15. Incubate in a humidified air incubator at 37 C with 5 % CO2.
Porous Membrane Culture Method for Expansion of Human Pluripotent Stem Cells
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16. The next day, change the medium to wash away any nonadherent cells. 17. Observe the cells daily using an inverted phase-contrast microscope. 18. Feed cells every 2 days by replacing the hASC medium. 19. Subculture cells using trypsin when hASCs reach 80–90 % confluence. 20. Split hASCs weekly at a 1:3 ratio. 3.2 Preparation of Feeder Cell on PM Insert (Scheme 1)
1. Pour 50 ml of hASC medium into a glass beaker. 2. Put the PM insert with an inverted status into a glass beaker and then put into a Teflon ring on the PM insert (see Note 6). 3. Remove extra hASC medium at the bottom of the PM insert. 4. After the preparation of PM insert, remove the hASC medium from a feeder plate. 5. Wash the plate twice with DPBS solution. 6. Dissociate the trypsinization.
hASCs
using
standard
methods
of
7. Incubate at 37 C incubator for 3 min. 8. Add 10 ml of hASC medium and pipette vigorously to disperse the hASC aggregates. 9. Count hASCs using a hemocytometer (see Note 7).
Scheme 1 Scheme for the preparation of feeder cells on PMs insert
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10. Centrifuge the counted cells at 300 g for 5 min. 11. Resuspend the cell pellet in 2 ml of hASC medium. 12. Gently pipette the cells few times up and down in the tube. 13. Seed hASCs onto the bottom of inverted PM at a density of 2 103 cells/cm2. 14. Incubate in a humidified air incubator at 37 C with 5 % CO2 for 24 h. 15. Remove the Teflon ring from the insert and observe hASCs at the bottom surface of the PM insert under a microscope. 16. Wash “the PM insert with hASCs feeder cells” using DPBS solution and move it to a 6-well plate. 3.3 hPSC Expansion on PM Insert (Scheme 2)
1. Prepare “the PM insert with hASCs feeder cells” as described above. 2. Add 5 ml of hPSC medium into each well containing the PM insert. 3. Transfer 60 hPSC clumps onto the PM insert using the mechanical transfer method (23) (see Note 8). 4. Label the 6-well plate with the hPSC line name, passage number, date, and your initials. 5. Place the plate into a humidified air incubator at 37 C with 5 % CO2 and then gently shake the plate (see Note 9). 6. After a 48-h incubation, observe hPSCs under a microscope. 7. Discard the spent medium with undetached cells and debris. 8. Gently add fresh hPSC culture medium to the PM insert. 9. Move the 6-well plate back into the humidified air incubator at 37 C with 5 % CO2.
Scheme 2 Scheme for hPSC maintain using proliferative feeder-based PMs insert culture systems
Porous Membrane Culture Method for Expansion of Human Pluripotent Stem Cells
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10. Refresh hPSC culture medium daily until cells require transfer. 11. After 6 total days of hPSCs culture, mechanically isolate fully grown hPSCs. 12. Passage hPSCs onto a new PM insert with fresh hASC feeder cells at a 1:5 ratio.
4
Notes 1. We advise using the same lot of FBS for the entire series of experiments. 2. We recommend preparing the cell stock solution on the same day and keeping it on ice. 3. Wash off the lipoaspirate extensively to remove most leukocytes and erythrocytes. If the medium from the final wash is still red, wash again by repeating steps 2–3. 4. We dissolve the required amount of collagenase in 50 ml of DPBS containing 1 % BSA and 2 mM CaCl2 and then filter sterilize it into the remaining working volume. Adipose tissue is shaken by hand every 10 min. 5. Be careful while handling cell pellets, as they are gelatinous and do not adhere readily to the centrifuge tubes. 6. Check that there is no medium leakage. This is important, because cells should attach to the bottom of PM with no significant loss. 7. For proliferative hASC feeder-based PM culture systems, we recommend a standard hASC plating density of 1–3 103 cells/cm2. A much higher initial feeder density may reduce the hPSC colony attachment rate. 8. This can be done by using a modified pipette, PIPETMAN tip, needle, or SweMed™ instrument. 9. We recommend to gently shake the plate side to side and back and forth to evenly distribute the cells. Avoid circular motions to prevent cell pooling in the center of the well.
Acknowledgment This work was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, and Future Planning (NRF-2013R-1A2A-1A09013980 and NRF-2012M3A9B4028569).
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