Isolation of Human Amnion Epithelial Cells According to Current Good Manufacturing Procedures

Roberto Gramignoli1, Raghuraman C. Srinivasan1, Kristina Kannisto1, Stephen C. Strom1

1 Department of Laboratory Medicine, Division of Pathology, Karolinska Institutet, Stockholm, Sweden
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1E.10
DOI:  10.1002/cpsc.2
Online Posting Date:  May, 2016
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Different cell types can be isolated from human placental tissues, and some have been reported to retain phenotypic plasticity and characteristics that make them a promising source of cells for regenerative medicine. Among these are human amnion epithelial cells (hAECs). Adoption of current good manufacturing practices (cGMP) and enhanced quality control is essential when isolating hAECs in order to deliver a safe and effective cellular product for clinical purposes. This unit describes a detailed protocol for selective isolation of hAECs from human term placenta with little to no contamination by other cell types. A method for characterizing the heterogeneity of the hAEC suspension is also provided. The resulting cell product will be useful for clinical as well as basic research applications. © 2016 by John Wiley & Sons, Inc.

Keywords: amnion; amnion epithelial cells; cGMP; placenta

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Table of Contents

  • Introduction
  • Basic Protocol 1: cGMP Protocol for Isolation of Human Amnion Epithelial cells
  • Support Protocol 1: Quality Assessment by flow Cytometry
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: cGMP Protocol for Isolation of Human Amnion Epithelial cells

  • Full‐term placenta, freshly delivered in sterile transport container
  • Plasmalyte solution (Baxter, cat. no. AFE0324D) or saline solution (9 mg/ml; ApoEx AB, cat. no. 259366)
  • Ringer's acetate solution (Baxter, cat. no. AFE0324D)
  • 10× TrypLE Select Enzyme (Life Technologies, cat. no. A12177‐01)
  • Trypan blue solution (Sigma‐Aldrich, cat. no. T8154)
  • BEL‐GEN cold storage solution (IGL Group, cat. no. BEL‐GEN/1000)
  • Dimethylsulfoxide (DMSO; WAK‐Chemie, cat. no. WAK‐DMSO10)
  • Laminar flow cabinet (BSL‐2)
  • Surgical drape
  • Sterile metal tray/basin
  • Sterile gloves
  • 2‐ml pipet, pyrogen free (VWR, cat. no. 414004‐264)
  • Sterile disposable scalpel (VWR, cat. no. SWAN0570)
  • Sterile steel scissors 18/10 (VWR, cat no. 233‐1237)
  • Sterile steel forceps (VWR, cat. no. 232‐0141)
  • 110‐ml sterile plastic container with lid (VWR, cat. no. 391‐0020)
  • 500‐ and 1000‐ml borosilicate glass beakers (VWR, cat. no. 213‐1126)
  • 50‐ml conical polypropylene tubes (Sigma‐Aldrich, cat. no. CLS430290)
  • Incubating rotator (e.g., Incubator‐Genie, Scientific Industries, cat. no. SI‐1402)
  • Burker chamber
  • 5‐ml cryogenic vials (VWR, cat. no. 479‐6845)
  • Controlled‐rate, alcohol‐free cell freezing container (e.g., CoolCell, Biocision, cat. no. BCS‐406)

Support Protocol 1: Quality Assessment by flow Cytometry

  • Directly conjugated antibodies:
  • Alexa Fluor 647–conjugated mouse anti‐human CD31 (IgG2; clone M89D3; Becton Dickinson, cat. no. 558094)
  • FITC–conjugated mouse anti‐human CD45 (IgG1; clone T29/33; Dako, cat. no. F0861)
  • APC–conjugated mouse anti‐human CD105 (IgG1; clone SN6; eBioscience, cat. no. 17‐1057)
  • PE–conjugated rat anti‐human CD49f (IgG1; clone GoH3; Becton Dickinson, cat. no. 555736)
  • FITC–conjugated mouse anti‐human CD326 (EpCAM; IgG1; clone HEA‐125; Miltenyi Biotec, cat. no. 130‐098‐113)
  • PE–conjugated mouse anti‐human glycoforin A (GlyA; IgG2; clone GAR‐2; Becton Dickinson, cat. no. 340947)
  • FITC–conjugated mouse IgG1 isotype control (Becton Dickinson, cat. no. 349041)
  • PE–conjugated rat IgG2 isotype control (Becton Dickinson, cat. no. 555844)
  • PE–conjugated mouse IgG2 isotype control (Becton Dickinson, cat. no. 349053)
  • APC–conjugated mouse IgG1 isotype control (Becton Dickinson, cat. no. 340442)
  • Alexa Fluor 647–conjugated mouse IgG2 isotype control (Becton Dickinson, cat. no. 564073)
  • Antibody diluent
  • Isolated hAECs (see protocol 1Basic Protocol)
  • Plasmalyte solution (Baxter, cat. no. AFE0324D) containing 5 mM EGTA (Sigma‐Aldrich, cat. no. E0396)
  • 5‐ml tubes for FACS analysis
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Literature Cited

Literature Cited
  Dominici, M., Le Blanc, K., Mueller, I., Slaper‐Cortenbach, I., Marini, F.C., Krause, D.S., Deans, R.J., Keating, A., Prockop, D.J., and Horwitz, E.M. 2006. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8:315‐317. doi: 10.1080/14653240600855905.
  Gramignoli, R., Marongiu, F., and Strom, S. 2016. Use of amnion epithelial cells in metabolic liver disorders. In: Placenta: The Tree of Life (O. Parolini, ed.), p. 143‐160. CRC Press, Taylor & Francis Group, New York.
  Hammer, A., Hutter, H., Blaschitz, A., Mahnert, W., Hartmann, M., Uchanska‐Ziegler, B., Ziegler, A., and Dohr, G. 1997. Amnion epithelial cells, in contrast to trophoblast cells, express all classical HLA class I molecules together with HLA‐G. Am. J. Reprod. Immunol. 37:161‐171. doi: 10.1111/j.1600‐0897.1997.tb00208.x.
  Kubo, M., Sonoda, Y., Muramatsu, R., and Usui, M. 2001. Immunogenicity of human amniotic membrane in experimental xenotransplantation. Invest. Ophthalmol. Vis. Sci. 42:1539‐1546.
  Miki, T. and S.C. Strom. 2006. Amnion‐derived pluripotent/multipotent stem cells. Stem Cell Rev. 2:133‐142. doi: 10.1007/s12015‐006‐0020‐0.
  Miki, T., Mitamura, K., Ross, M.A., Stolz, D.B., and Strom, S.C. 2007. Identification of stem cell marker‐positive cells by immunofluorescence in term human amnion. J. Reprod. Immunol. 75:91‐96. doi: 10.1016/j.jri.2007.03.017.
  Miki, T., Marongiu, F., Dorko, K., Ellis, E.C.S., and Strom, S.C. 2010. Isolation of amniotic epithelial stem cells. Curr. Protoc. Stem Cell Biol. 12:1E.3.1‐1E.3.10.
  Skvorak, K.J., Dorko, K., Marongiu, F., Tahan, V., Hansel, M.C., Gramignoli, R., Gibson, K.M., and Strom, S.C. 2013a. Placental stem cell correction of murine intermediate maple syrup urine disease. Hepatology 57:1017‐1023. doi: 10.1002/hep.26150.
  Skvorak, K.J., Dorko, K., Marongiu, F., Tahan, V., Hansel, M.C., Gramignoli, R., Arning, E., Bottiglieri, T., Gibson, K.M., and Strom S.C. 2013b. Improved amino acid, bioenergetic metabolite and neurotransmitter profiles following human amnion epithelial cell transplant in intermediate maple syrup urine disease mice. Mol. Genet. Metab. 109:132‐138. doi: 10.1016/j.ymgme.2013.02.011.
  Strom, S.C., Skvorak, K., Gramignoli, R., Marongiu, F., and Miki, T. 2013. Translation of amnion stem cells to the clinic. Stem Cells Dev. 22 Suppl 1:96‐102. doi: 10.1089/scd.2013.0391.
  Takashima, S., Yasuo, M., Sanzen, N., Sekiguchi, K., Okabe, M., Yoshida, T., Toda, A., and Nikaido, T. 2008. Characterization of laminin isoforms in human amnion. Tissue Cell 40:75‐81. doi: 10.1016/j.tice.2007.09.001.
  Wolbank, S., Peterbauer, A., Fahrner, M., Hennerbichler, S., van Griensven, M., Stadler, G., Redl, H., and Gabriel, C. 2007. Dose‐dependent immunomodulatory effect of human stem cells from amniotic membrane: A comparison with human mesenchymal stem cells from adipose tissue. Tissue Eng. 13:1173‐1183. doi: 10.1089/ten.2006.0313.
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