Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 May 30;11(1):87-95.
doi: 10.15283/ijsc18001.

Large-scale Isolation, Expansion and Characterization of Human Amniotic Epithelial Cells

Sanjay Gottipamula  1 , K N Sridhar  1
Affiliations

Large-scale Isolation, Expansion and Characterization of Human Amniotic Epithelial Cells

Sanjay Gottipamula et al. Int J Stem Cells. .

Abstract

Background and objectives: The human Amniotic epithelial cells (AME) derived from amniotic membrane of placenta have been considered as the potential fetal stem cell source with minimal or no ethical concerns and are important therapeutic tool for anti-fibrotic and regenerative therapies.

Methods and results: Here, we evaluated the isolation, media screening, scale-up and characterization of AME cells. The isolation, expansion of AMEs were performed by sequential passaging and growth kinetics studies. The AMEs were characterized using immunocytochemistry, immunophenotyping, In-vitro differentiation, and anti-fibrotic assays. The growth kinetics study revealed that the AME cultured in Ultraculture (UC) and DMEM knockout (DMEM-KO) have prominently higher growth rate compared to others. Overall, the AMEs cultured from 5 different media retained basic morphological characteristics and the functional characteristics.

Conclusions: Our result suggests that the AMEs can be successfully cultured in UC based complete media without losing its epithelial cell characteristics even after passaging for passage 2 (P2). However, a careful and methodical pre-clinical and clinical translation studies need to be conducted to show its safety and efficacy.

Keywords: Amniotic epithelial cells; Cell therapy; Cryopreservation; Growth kinetics; Stability; Tissue engineering.

PubMed Disclaimer

Conflict of interest statement

Potential Conflict of Interest

The authors have no conflicting financial interest.

Figures

Fig. 1
Fig. 1
Growth Kinetics of AME sub cultured in 5 different media. (A) Cumulative population doublings of AMEs plotted against the passage number. (B) Population doubling time of AMEs (Epi is significantly higher (***p<0.001) across all passages compared to all other media, except Epi vs KOSR showed *p<0.05).
Fig. 2
Fig. 2
Immunophenotyping of AME at P1. A representative histogram of isotypes white and grey as EpCAM (A) and Cyto (B) (Cytokeratin 4, 5, 6, 10, 13, 18) of AME cultured in UC respectively. (C) Overall bar chart of Immunophenotyping of EpCAM and cyto expression of AME in all 5 different cell culture media.
Fig. 3
Fig. 3
In-vitro differentiation of AME: (A, C, E) and (G) are un-induced AME as controls and (B, D, F) and (H) are induced with respective induction media. Scale bar – 100 microns.
Fig. 4
Fig. 4
Conditioned media mediated fibroblast inhibition assay: Conditioned media of AME, BME and controls were added to fibroblast culture and incubated for 24 h before the proliferation was assessed via MTT assay. Both AME and BME conditioned media are able to inhibit the fibroblast proliferation at two different cell dose of 50K and 25K. Data are normalized to mean proliferation of fibroblast alone as 100% (n=3, pooled data from 3 separate experiments).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Mason C, Brindley DA, Culme-Seymour EJ, Davie NL. Cell therapy industry: billion dollar global business with unlimited potential. Regen Med. 2011;6:265–272. - PubMed
    1. You HJ, Han SK. Cell therapy for wound healing. J Korean Med Sci. 2014;29:311–319. - PMC - PubMed
    1. Varkey M, Ding J, Tredget EE. Advances in skin substitutes-potential of tissue engineered skin for facilitating anti-fibrotic healing. J Funct Biomater. 2015;6:547–563. - PMC - PubMed
    1. Dodson BP, Levine AD. Challenges in the translation and commercialization of cell therapies. BMC Biotechnol. 2015;15:70. - PMC - PubMed
    1. Ackermann K, Borgia SL, Korting HC, Mewes KR, Schäfer-Korting M. The Phenion full-thickness skin model for percutaneous absorption testing. Skin Pharmacol Physiol. 2010;23:105–112. - PubMed

LinkOut - more resources