Stem Cell Culture and Expansion PDF

STEM CELL Stem cell biotechnolgy CULTURE AND EXPANSION Nguyễn Minh Triết – 20187211 1. INTRODUCTION 2 1. INTRODUCTION Human embryonic stem cells (hESCs) are generally maintained on a layer of inactive murine embryonic fibroblast (MEF) feeder cells. hESCs can be ma...

STEM CELL Stem cell biotechnolgy CULTURE AND EXPANSION Nguyễn Minh Triết – 20187211 1. INTRODUCTION 2 1. INTRODUCTION Human embryonic stem cells (hESCs) are generally maintained on a layer of inactive murine embryonic fibroblast (MEF) feeder cells. hESCs can be maintained under these conditions for several passages without compromising the cells’ proliferation or pluripotency and differentiation potential 3 1. INTRODUCTION Induced pluripotent stem cells (iPSCs) are genetically reprogrammed adult cells that exhibit a pluripotent stem cell-like state similar to embryonic stem cells. While these artificially generated cells are not known to exist in the human body, they show qualities remarkably similar to those of embryonic stem cells (ESCs); thus, they are an invaluable new source of pluripotent cells for drug discovery, cell therapy, and basic research. 4 1. INTRODUCTION Somatic (adult) stem cells are found in most major organs and tissues, and are currently being isolated from many tissues in the body. Mesenchymal stem cells (MSCs) were originally isolated from bone marrow stroma but were subsequently found to be present in most tissues of the body including cord blood, adipose tissue, skin 5 2. CULTURING AND EXPANSION OF EMBRYO STEM CELL AND INDUCED PLURIPOTENT STEM CELL 6 1. ISOLATION OF ECS AND iPSC 7  CytoTune®-iPS 1. ISOLATION Sendai Reprogramming System uses vectors based on replication in competent Sendai virus (SeV) to safely and effectively deliver and express key genetic factors necessary for reprogramming somatic cells into iPSCs. The CytoTune®-iPS Sendai Reprogramming Kit contains four SeV-based reprogramming 8 2. PREPARE AND CULTURE ECS AND iPSC 9 2. PREPARE AND CULTURE ECS AND iPSC hESCs cultured on mitotically iPSCs cultured on mitotically inactivated murine embryonic inactivated MEF feeder layer in fibroblast (MEF) feeder layer in PSC Culture Medium containing 10 PSC Culture Medium containing KSR 3. THAWING ECS AND IPCS PSC culture 11 4. PASSAGING ECS AND iPCS Colonies ready to be passaged. The large colony and the close proximity of the colonies to each 12 4. PASSAGING ECS AND iPCS 13 4. PASSAGING ECS AND iPSC Colony pulling away from iMEF Colony after being cut with the layer after treatment with enzyme StemPro® EZPassage™ Disposable Cell Passaging Tool 14 2. CULTURING AND EXPANSION OF MESENCHYMAL STEM CELL 15 1. ISOLATION OF MCS 16  2. CULTURING AND EXPANSION + Diabetes medication or insulin therapy. Transfer the centrifuge at cells to a 15- 210 × g for 5 Determine Add a minutes the total incubate at Incubate at ml conical Add Rinse the sufficient Add the number of 37℃, 5% 37℃, 5% tube MesenPRO surface of the volume of equivalent of 2 cells CO2 and 90% CO2 and 90% cell layer with prewarme volumes RS™ Medium humidity humidity DPBS d TrypLE MesenPRO to each RS™ Medium. vessel 17 THANK YOU FOR LISTENING!