By Jeanne F. Loring Ed., Suzanne Peterson, Jeanne F. Loring, Robin L. Wesselschmidt, Philip H. Schwartz
Stem cells are self-replicating and undifferentiated, which means their functionality isn't but telephone, tissue, or organ-specific. as a result targeted nature of those cells, learn into their biology and serve as holds nice promise for healing functions via substitute or fix of diseased and broken cells. This reader-friendly guide presents a realistic "hands on" advisor to the tradition of human embryonic and somatic stem cells. through offering equipment for embryonic and grownup strains side-by-side, the authors lay out a chic and precise route to knowing the technology of stem telephone perform. The authors commence with a broad-based advent to the sector, and likewise overview felony and regulatory matters and patents. every one experimental technique is gifted with an ancient creation, distinctive strategy, dialogue of other tools, and customary pitfalls. This lab advisor for researchers additionally serves as a textbook for undergraduate and graduate scholars in laboratory classes.
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Additional resources for Human Stem Cell Manual: A Laboratory Guide
2), but a higher proportion of differentiated cells must be expected. Passaging hESCs hESCs, unlike mouse ESCs, do not survive well when dissociated to single cells. Therefore, the most reliable method for passaging undifferentiated hESC cultures is manual dissection of the colonies. This method may seem tedious, but it is virtually foolproof and we recommend that novices use this method until they have familiarity with the cells and can easily recognize differentiation in the culture. We also recommend manual passaging for producing cell banks of low-passage hESCs.
29 30 Human Stem Cell Manual 3. Store frozen at Ϫ20°C or Ϫ80°C for long-term storage. 4. Thawed aliquots are stable for up to two weeks at 4°C. Stock solutions Collagenase IV (200 U/mL) Component Amount Stock concentration Collagenase IV 20 000 units 200 units/mL 1. Dissolve 20 000 units in 100 mL of knockout D-MEM. 2. Filter (2 μm). Aliquot and store at Ϫ20°C. 3. Alternatively, make 1 mg/mL solution, filter, aliquot, and store at Ϫ20°C. MEF medium (for propagation of MEFs) (500 mL) Component Amount Final concentration D-MEM, high glucose FBS Non-essential amino acids (100ϫ) L-Glutamine (200 mM) Pen-strepa (100ϫ) 435 mL 50 mL 5 mL 5 mL 5 mL 90% 10% 1ϫ 2 mM 1ϫ a Antibiotics optional.
Try to achieve a relatively uniform suspension of cell clumps containing several hundred cells each. 7. The cells can be split among 3–6 dishes of the same size as the original culture, depending on density of the original culture. If you wish to put the cells in different sized dishes, calculate the dilution based on surface area of each type of dish. Non-enzymatic cell dissociation Ca2ϩ- and Mg2ϩ-free saline solutions containing EDTA or EGTA have not been as widely used for hESC dissociation as the methods described above, but they should offer advantages for assays that require intact cell surface proteins such as flow cytometry and immunocytochemistry.