再生醫學為目前醫療的新趨勢,FDA已核准三例胚胎幹細胞的人體試驗.幹細胞要進入人體,培養上需安全無疑與穩定,才能放心進行移植.
傳統的幹細胞培養是將細胞培養在一層滋養層(feeder cells)上,滋養層主要為老鼠的纖維母細胞(mouse fibroblasts),並加入含胎牛血清的培養液.因為胎牛血清含牛的白蛋白,生長激素,抗體,甚至病毒,對人體細胞來說是外來的抗原,可能產生排斥的過敏反應和病毒感染的可能.
美國加州大學洛杉磯分校(UCLA)的幹細胞研究團隊日前發表研究成果,利用三個小分子抑制物(3 small molecule inhibitors)與纖維母細胞生長因子(bFGF;basic fibroblast growth factor)的組合,成功讓幹細胞能長期在無滋養層與無動物血清培養液下生長.
研究人員利用細胞的負回饋機制(feedback system scheme)找出適當的抑制物與濃度,利用含抑制物的人工培養液不僅讓幹細胞可以穩定生長,並且同時抑制病毒的感染與癌化的產生.這個適用於臨床級的培養液已開始接受優良製造規範(good manufacturing practice)和美國食品藥物管理局(Food and Drug Administration)的審核與評估.而這項發現已在25日刊登在Nature Communications期刊中.
An optimized small molecule inhibitor cocktail supports long-term maintenance of human embryonic stem cells
In regenerative medicine, large supplies of safe and reliable human embryonic stem (hES) cells are needed for implantation into patients, but the field has faced challenges in developing cultures that can consistently grow and maintain clinical-grade stem cells.
Standard culture systems use mouse "feeder" cells and media containing bovine sera to cultivate and maintain hES cells, but such animal product–based media can contaminate the cells. And because of difficulties in precise quality control, each batch of the medium can introduce new and unwanted variations.
Now, a team of stem cell biologists and engineers from UCLA has identified an optimal combination and concentration of small-molecule inhibitors to support the long-term quality and maintenance of hES cells in feeder-free and serum-free conditions. The researchers used a feedback system control (FSC) scheme to innovatively and efficiently select the small-molecule inhibitors from a very large pool of possibilities.
The research findings, published Jan. 25 in the journal Nature Communications, represent a major advance in the quest to broadly transition regenerative medicine from the benchtop to the clinic.
"What is significant about this work is that we've been able to very rapidly develop a chemically defined culture medium to replace serum and feeders for cultivating clinical-grade hES cells, thereby removing a major roadblock in the area of regenerative medicine," said Chih-Ming Ho, the Ben Rich–Lockheed Martin Professor at the UCLA Henry Samueli School of Engineering and Applied Science and a member of the National Academy of Engineering.
Unlike current animal product–based media, the new medium is a "defined" culture medium — one in which every component is known and traceable. This is important for clinical applications and as drugs or cells enter the world of regulatory affairs, including good manufacturing practice compliance and Food and Drug Administration supervision.
"It is also the first defined medium to allow for long term single-cell passage," said the paper's senior author, Hong Wu, the David Geffen Professor of Molecular and Medical Pharmacology at the David Geffen School of Medicine at UCLA and a researcher with UCLA's Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research.
Single-cell passaging — a process in which hES cells are dissociated into single cells and subcultured through single-cell–derived colonies — is important in overcoming the massive cell death associated with hES cell dissociation during routine passage, and it allows for genetic manipulation at the clonal level.
Tsutsui, then a UCLA Engineering graduate student, and Bahram Valamehr, then a graduate student at the Geffen School of Medicine, started working on the project two years ago. Armed with biological readouts and analyses of stem cells mastered in Hong Wu's laboratory through the lab's extensive accomplishments in stem cell research, Tsutsui and Valamehr used the FSC scheme — developed previously by Ho's group to search for optimal drug combinations for viral infection inhibition and cancer eradication — to facilitate the rapid screening of a very large number of possibilities.
