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Culturing with modified EGM 2 medium enhances porcine neonatal islet‐like cell clusters resistance to apoptosis in islet xenotransplantation
Author(s) -
Ma Xiaoqian,
Yang Cejun,
Zhang Juan,
Wang Jia,
Li Wei,
Xu Chang,
Rong Pengfei,
Ye Bin,
Wu Minghua,
Jiang Jianhui,
Yi Shounan,
Wang Wei
Publication year - 2017
Publication title -
xenotransplantation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.052
H-Index - 61
eISSN - 1399-3089
pISSN - 0908-665X
DOI - 10.1111/xen.12358
Subject(s) - xenotransplantation , islet , apoptosis , microbiology and biotechnology , andrology , transplantation , immunology , biology , medicine , endocrinology , diabetes mellitus , biochemistry
Background Neonatal pig islet‐like cell clusters ( NICC ) are an attractive source of insulin‐producing tissue for potential transplantation treatment of type 1 diabetic patients. However, a considerable loss of NICC after their transplantation due to apoptosis resulted from islet isolation and instant blood‐mediated inflammatory reaction remains to be overcome. Methods EGM 2 medium depleted with hydrocortisone and supplemented with 50 mmol/L isobutylmethylxanthine, 10 mmol/L nicotinamide, and 10 mmol/L glucose was used to culture NICC at day 1, the day after isolation and changed every other day. NICC cultured with EGM 2 or control Ham's F‐10 medium were collected at day 7 of culture for the following assays. The viability of NICC was evaluated by AO / EB staining and FACS . Static assay and oxygen consumption rate analysis were performed to assess the function of NICC . Insulin and glucagon gene expression were measured by real‐time PCR . Tubing loops model and TUNEL assay were performed to confirm the apoptosis‐resistant ability of NICC cultured with modified EGM 2 medium. Serum starvation and hypoxia treatment were used to test the tolerant capability of NICC in the microenvironment of hypoxia/nutrient deficiency in vitro. The molecules involved in apoptosis pathways in NICC were analyzed by Western blotting. Results Compared with Ham's F‐10 medium, culturing NICC with EGM 2 medium led to increased number and viability of NICC with higher stimulation index, upregulated gene expression of both insulin and glucagon, and enhanced mitochondria function. Furthermore, fewer modified EGM 2 medium cultured NICC were found under apoptosis when evaluated in an in vitro tubing loop model of IBMIR . Moreover, EGM 2 medium cultured NICC demonstrated much less apoptotic cells under either serum starvation or hypoxia condition than their Ham's F‐10 medium cultured counterparts. The enhanced capability of EGM 2 cultured NICC to resist apoptosis was associated with their elevated protein levels of anti‐apoptotic Bcl‐2 family member Mcl‐1. Conclusion Culturing NICC with EGM 2 provides a simple and effective approach not only to increase NICC yield, viability, and maturation but also to enhance their resistance to apoptosis to preserve the initial graft mass for successful islet xenotransplantation.