Establishment of Human Feeder Cell Lines

1. Individually wrapped sterile universal containers for collection and transport of biopsies (Sarstedt, Ingle Farm, Australia; cat. no. 75.9922-721).

2. Earle's or Hank's balanced salt solution (100 mL, Sigma Chemical Co, St. Louis, MO; cat. no. H9269) supplemented with 1 mL penicillin-streptomycin solution (Invitrogen, Carlsbad, CA; cat. no. 15070-063).

3. hES medium: 50% Dulbecco's modified Eagle's medium (Invitrogen; cat. no. 11960-044), 50% human serum (HS) (Irvine Scientific, Santa Ana; cat. no. 5009), 2 mM l-glutamine (Invitrogen; cat. no. 15070-063), 1% nonessential amino acids (Invitrogen; cat. no. 11140-050), 0.1 mM 2-mercaptoethanol (Invitrogen; cat. no. 21985-023), 50 |L/mL penicillin/50 |g/mL streptomycin solution (Invitrogen; cat. no. 15070-063 (see Note 1).

4. 0.05% trypsin-0.53 M ethylenediaminetetraacetic acid (EDTA) (100 mL, Invitrogen; cat. no. 25300-062).

5. Plastic cell culture flasks (BD BioSciences, Bedford, MA; cat. no. 353018) or one-well culture dishes (BD Falcon; cat. no. 353653).

6. Two pairs of sterile watchmaker forceps (Lawton, Fridingen, Germany; cat. no. 09-0959).

7. One pair of pointed, curved scissors (Lawton; cat. no. 05-0311).

8. 1-mL, 5-mL sterile serological pipets (Becton Dickinson [BD], Franklin Lakes, NJ; cat. no. 7543, 7521).

9. 5-mL, 10-mL sterile plastic tubes (BD; cat. no. 357553, 357551).

10. Sterile cell scraper (Nunclon, Roskilde, Denmark; cat. no. 179693).

2.2. Freezing of Human Feeder Cells

1. Cell culture freezing medium with 10% dimethylsulfoxide as cryoprotectant (Invitrogen; cat. no. 11101-011).

5. 5-mL, 10-mL sterile plastic tubes (BD Falcon, Bedford, MA; cat. no. 2003 and 2001).

6. Programmable freezing machine (Planer, Stanbury UK; Series III).

2.3. Thawing of Human Feeder Cells

2. 5-mL, 10-mL sterile plastic tubes (Falcon; cat. no. 2003 and 2001).

3. Plastic cell culture flasks (BD Falcon; cat. no. 353018).

3. Methods

3.1. Establishment of Human Feeder Cell Lines

1. Aseptically collect either single or multiple 4 mm2 adult skin biopsies from the abdominal regions of adult donors or similar sized fetal muscle biopsies from the thigh regions of 14-wk-old normal human abortuses.

2. Transfer biopsies to a sterile Petri dish containing hES medium.

3. For adult skin, using sterile watchmaker forceps and pointed scissors, separate the dermis from epidermis and cut the epidermis into very tiny explants (1 mm3). For fetal muscle, using sterile pointed scissors, mince the tissue into tiny pieces or clusters of cells.

4. Transfer the epidermal explants or fetal muscle tissue pieces to fresh hES medium in another sterile Petri dish. Rinse explants in this medium several times.

5. Transfer the explants to small cell culture flasks or one-well culture dishes containing a layer of hES medium enough only to completely immerse and bathe the explants (see Note 2). Incubate flasks/dishes at 37°C in 5% CO2 in air and do not disturb/shake the cultures for at least 3 d (see Note 3).

6. Examine cultures after 3 d under phase contrast-inverted optics for cellular outgrowths and to see if medium has become straw colored. Whether there are outgrowths or not, add an equal amount of fresh hES medium to the existing medium and incubate further (see Note 4). If there is further growth of outgrowths from explants after 2 d of incubation, keep replacing medium with fresh hES medium until confluency or substantial growth from the explants are obtained. Epidermal outgrowths from adult skin start their primary growth as epithelioid islands

Primary Explant And Outgrowth
Fig. 1. Light micrograph of an island of epithelioid outgrowth from a primary explant of human adult epidermal skin (x200). (Please see the companion CD for the color version of this figure.)

(Fig. 1). After the first passage, the epithelial cells transform into fibroblasts (Fig. 2). Fetal muscle explants commence their primary growth as fibroblasts and remains as fibroblasts thereafter in subsequent passages.

7. For passaging, aspirate medium and discard. Add 2 mL 0.05% trypsin-0.53 mM EDTA. Incubate for a few minutes, checking each time whether the monolayer has detached from plastic (see Note 5). After explants and individual cells have detached, transfer entire contents to sterile 5- or 10-mL plastic tubes and centrifuge at 300g for 5-10 min (see Note 6).

8. Discard supernatant. Add 2 mL hES medium to the pellet. Resuspend and seed cell suspension into new cell culture flasks/one-well dishes containing hES medium. Incubate and do not disturb for 2 d. For passaged cells, faster growth with earlier confluency is expected. The cells can now be frozen for later use as support for hES cells (see Note 7).

3.2. Freezing of Human Feeder Cells (see Note 8)

1. Monolayers are washed and trypsinized (similar to passaging fresh cells described in Subheading 3.1., step 7) to allow detachment from plastic.

Fondo Red Pescar Tumblr
Fig. 2. Light micrograph of epithelioid outgrowths transforming into fibroblasts in early passages of human adult epidermal skin cultures (x200). (Please see the companion CD for the color version of this figure.)

2. The trypsinized cells are transferred to 5-mL tubes and washed twice with hES medium by centrifugation to remove all traces of trypsin.

3. The washed cell pellet is resuspended in 2 mL of freezing medium and approx 2-3 million cells are seeded into each cryotube, which is labeled with passage number, cell type, and date of freezing.

4. The cryotubes are frozen using the slow-programmed freezing machine. Cooling is from room temperature to -30°C at 1°C per minute. At -30°C, the cryotubes are plunged directly into liquid nitrogen.

3.3. Thawing of Human Feeder Cells

1. The cryotubes are warmed at 37°C using a water bath and the cells are transferred to a sterile 10-mL Falcon tube.

2. About 9 mL of culture medium is added slowly to the cells drop by drop to prevent osmotic shock to the cells and the cell suspension is centrifuged at 300g for 5 min.

3. The supernatant is removed and the pellet is resuspended in 1 mL hES medium.

4. The cell suspension is then seeded into two to three cell culture flasks, each containing 2-3 mL of hES medium, and the flasks are incubated at 37°C in 5% CO2 in air. Growth to confluency is monitored.

4. Notes

1. If a xeno-free in vitro system is not mandatory, Chang's medium (Irvine Scientific; cat. no. C106) supplemented with 200 mM l-glutamine, streptomycin (100 |g/mL), and Fungizone (0.1 mL/mg) (Invitrogen; cat. no. 15290-018) is a better substitute for hES medium + 50% HS. Chang's medium is an enriched super-complex commercial medium that contains salts, energy substrates, newborn calf serum, amino acids, hormones, and vitamins. Good cell growth and shorter times to monolayer confluency are produced with Chang's medium (7,8).

2. If a cell culture flask is used, loosen cap.

3. The cultures are not disturbed for 3 d to allow the explants to attach to the plastic and produce outgrowths. This is usually the point of failure if one is not careful. The medium is adequate for 3 d and does not need changing.

4. Do not change old medium, because the attached explants will be disturbed. It is common to see some explants floating without attachment. After 2 d of incubation, if there is no growth, there is a likely possibility of contamination.

5. Detachment can be hastened by mechanical agitation.

6. If it is necessary to avoid trypsin, the cells can be easily detached mechanically with a sterile cell scraper. The detached sheets of cells need to be broken into cell clusters or individual cells by vigorous resuspension using a sterile Pasteur pipet.

7. For primary cultures and early passages, use hES medium supplemented with 50% HS; for subsequent passages, use hES medium supplemented with 20% HS.

8. Maintenance of live cells in the incubator (37°C) can be very labor intensive and costly. Thus cells are frozen in liquid nitrogen for future use without the need for frequent passaging.

References

1 Bongso, A., Fong, C. Y., Ng, S. C., and Ratnam, S. S. (1994) The growth of inner cell mass cells from human blastocysts. Theriogenology 41, 161.

2 Bongso, A., Fong, C. Y., Ng, S. C., and Ratnam, S. S. (1994) Isolation and culture of inner cell mass cells from human blastocysts. Hum. Reprod. 9, 2110-2117.

3 Richards, M., Fong, C. Y., Chan, W. K., Wong, P. C., and Bongso, A. (2002) Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat. Biotech. 20, 1-4.

4 Richards, M., Tan, S., Fong, C. Y., Biswas, A., Chan, W. K., and Bongso, A. (2003) Comparative evaluation of various human feeders for prolonged undifferentiated growth of human embryonic stem cells. Stem Cells 21, 546-556.

5 Xu, C., Inokuma, M. S., and Denham, J. (2001) Feeder-free growth of undifferentiated human embryonic stem cells. Nat. Biotech. 19, 971-974.

6. Draper, J. S., Smith, K., Gokhale, P., et al. (2003) Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nature Biotech. Online 2, 53-54.

7. Bongso, A., Ng, S. C., Sathananthan, A. H., Ng, P. L., Rauff, M., and Ratnam, S. S. (1989) Establishment of human ampullary cell cultures. Hum. Reprod. 4, 486-490.

8. Bongso, A., Gajra, B., Ng, P. L., Wong, P. C., Ng, S. C., and Ratnam, S. S. (1988) Establishment of human endometrial cell cultures. Hum. Reprod. 3, 705-710.

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