Lam Alan Tin Lun, Ho Valerie, Vassilev Svetlan, Reuveny Shaul, Oh Steve Kah Weng
Stem Cell Bioprocessing, Bioprocessing Technology Institute, Agency for Science, Technology and Research, Singapore, Republic of Singapore.
Cell Prolif. 2022 Aug;55(8):e13256. doi: 10.1111/cpr.13256. Epub 2022 May 19.
Induced pluripotent stem cells (iPSCs) generated by monolayer cultures is plagued by low efficiencies, high levels of manipulation and operator unpredictability. We have developed a platform, reprogramming, expansion, and differentiation on Microcarriers, to solve these challenges.
Five sources of human somatic cells were reprogrammed, selected, expanded and differentiated in microcarriers suspension cultures.
Improvement of transduction efficiencies up to 2 times was observed. Accelerated reprogramming in microcarrier cultures was 7 days faster than monolayer, providing between 30 and 50-fold more clones to choose from fibroblasts, peripheral blood mononuclear cells, T cells and CD34+ stem cells. This was observed to be due to an earlier induction of genes (β-catenin, E-cadherin and EpCAM) on day 4 versus monolayer cultures which occurred on days 14 or later. Following that, faster induction and earlier stabilization of pluripotency genes occurred during the maturation phase of reprogramming. Integrated expansion without trypsinization and efficient differentiation, without embryoid bodies formation, to the three germ-layers, cardiomyocytes and haematopoietic stem cells were further demonstrated.
Our method can solve the inherent problems of conventional monolayer cultures. It is highly efficient, cell dissociation free, can be operated with lower labor, and allows testing of differentiation efficiency without trypsinization and generation of embryoid bodies. It is also amenable to automation for processing more samples in a small footprint, alleviating many challenges of manual monolayer selection.
单层培养产生的诱导多能干细胞（iPSC）存在效率低、操作复杂且操作人员难以预测等问题。我们开发了一个微载体上重编程、扩增和分化的平台来解决这些挑战。
在微载体悬浮培养中对五种人类体细胞来源进行重编程、筛选、扩增和分化。
观察到转导效率提高了2倍。微载体培养中的重编程加速，比单层培养快7天，从成纤维细胞、外周血单核细胞、T细胞和CD34 +干细胞中获得的克隆数量多出30至50倍。这被认为是由于在第4天比单层培养更早地诱导了基因（β-连环蛋白、E-钙黏蛋白和EpCAM），单层培养在第14天或更晚才出现这种情况。在此之后，在重编程的成熟阶段多能性基因的诱导更快且稳定更早。进一步证明了无需胰蛋白酶消化即可进行整合扩增，并且能够高效分化为三个胚层、心肌细胞和造血干细胞，而无需形成胚状体。
我们的方法可以解决传统单层培养的固有问题。它效率高、无需细胞解离、操作劳动强度低，并且无需胰蛋白酶消化和形成胚状体即可测试分化效率。它还适合自动化操作，可在小空间内处理更多样本，减轻了手动单层筛选的许多挑战。
