Storable bFGF-Releasing Membrane Allowing Continuous Human iPSC Culture

Abstract

Basic fibroblast growth factor (bFGF) is a crucial supplement for culture media of human pluripotent stem cells. However, bFGF is extremely unstable under cell culture conditions, which makes frequent (generally every day) medium refreshment requisite. We recently developed a water-floatable, bFGF-releasing membrane via a simple bFGF adsorption process following oxygen plasma treatment by utilizing a polyethylene nonwoven fabric as an adsorbent. This membrane allowed sustained release of bFGF while floating on medium, thereby keeping the bFGF concentration in the medium sufficient for maintaining human-induced pluripotent stem cells (iPSCs) in a proliferative and pluripotent state for as long as 3 days. In this study, lyophilization was applied to the membrane to stabilize bFGF. The sustained bFGF-releasing function of the membrane was kept unchanged even after lyophilization and subsequent cryopreservation at −30 °C for 3 months. The cryopreserved membrane supported proliferation and colony formation of human iPSCs while retaining their viability and pluripotency in a medium-change-free continuous culture for 3 days. The present bFGF-releasing membrane is ready-to-use, storable for at least 3 months, and obviates daily medium refreshment. Therefore, it is a new and more practical bFGF supplement for culture media of human stem cells.

Design of chemically defined synthetic substrate surfaces for the in vitro maintenance of human pluripotent stem cells: A review

“…Then, much effort has been involved to identify the essential markers relating to the pluripotency maintenance of hPSCs. In most researches, markers include POU5F1, NANOG, SOX2, glycolipid stage specific embryonic antigen 3 (SSEA3), SSEA4, REX‐1, tumor rejection antigen 1‐60 (TRA‐1 60) and TRA‐1 81 were commonly used 19–22 . In 2018, Yilmaz et al identified a set of 50 genes essential for the normal growth and survival of hPSCs using CRISPR‐Cas9 based genome‐wide screens 23 .…”

“…Apart from the substitution of growth factors in medium, researchers focus on new formulations of the factors, especially for unstable and quick lose bFGF during the cell culture. bFGF‐encapsulated biodegradable polymer microbeads, multi‐trilayered nanofilms composed of repeating polycation/polyanion/bFGF units and bFGF absorption polyethylene nonwoven fabric have developed for the sustain release of bFGF 21,45,46 . Notably, only the last nonwoven fabric material used a bFGF‐free medium and successfully maintained the culture of hPSCs for 15 days 46 .…”

“…In most researches, markers include POU5F1, NANOG, SOX2, glycolipid stage specific embryonic antigen 3 (SSEA3), SSEA4, REX-1, tumor rejection antigen 1-60 (TRA-1 60) and TRA-1 81 were commonly used. [19][20][21][22] In 2018, Yilmaz et al identified a set of 50 genes essential for the normal growth and survival of hPSCs using CRISPR-Cas9 based genome-wide screens. 23 In 2020, Ghosh et al…”

“…bFGFencapsulated biodegradable polymer microbeads, multi-trilayered nanofilms composed of repeating polycation/polyanion/bFGF units and bFGF absorption polyethylene nonwoven fabric have developed for the sustain release of bFGF. 21,45,46 Notably, only the last nonwoven fabric material used a bFGF-free medium and successfully maintained the culture of hPSCs for 15 days. 46 Recently, in 2020, Khalil et al developed a mineral-coated microparticles to load bFGF, which can support the self-renew of hPSCs over 25 passages in bFGF-free medium.…”

Induction of angiogenesis and neural progenitor cells by basic fibroblast growth factor‐releasing polyglycolic acid sheet following focal cerebral infarction in mice

“…Recently, bFGF‐releasing sheets were prepared from polyethylene nonwoven fabric, via oxygen plasma treatment followed by bFGF adsorption without using animal‐derived additives 15,16 . The adsorbed bFGF is released into a culture medium in a sustained manner, maintaining the bFGF concentration in the medium at ≥10 ng/ml for 3 days 15 .…”