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Vancouver, British Columbia, Canada, 2013/08/26 - STEMCELL Technologies offers mTeSR™1 (defined) and TeSR™2 (animal protein-free and defined) maintenance medium for feeder-independent cultures of embryonic stem (ES) and induced pluripotent stem (iPS) cells - Stemcell.com.
Some of the critical aspects of maintaining healthy ES and iPS cell cultures are outlined in the points below.
1. Appropriate matrix
Using the right matrix is critical to successful feeder-free cultures of ES and iPS cells in mTeSR™1 and TeSR™2. We recommend BD Matrigel™ hESC-qualified Matrix (BD, Catalog #354277) or Vitronectin XF™ (Catalog #07190) for mTeSR™1 cultures, and StemAdhere™ Defined Matrix for PSC (Catalog #07160) for TeSR™2 cultures. Always follow the supplier’s instructions when storing, diluting, and coating the plates with the matrices. Cells will fail to attach if plates are not coated appropriately with the right matrices. For information on how to coat plates properly with Matrigel™, please refer to our technical tip here.
2. Timing between successive passages
Using the same size and density of seeded aggregates, cells cultured in TeSR™2 grow slightly faster than those grown in mTeSR™1. Therefore mTeSR™1 cultures are usually passaged every 5-7 days, whereas TeSR™2 cultures are passaged every 4-6 days (dependent on the cell line). There is an approximate 24 hour window that is optimal for passaging cells grown in mTeSR™1 or TeSR™2. Cultures should be passaged when there are large colonies, with dense centers, and the colonies are just beginning to merge (i.e. plate is approximately 60 - 75% confluent).
3. Removing areas of differentiation
Before passaging, areas of differentiation should be selectively removed by scraping or aspirating off. If not removed, the differentiated cells will overgrow, and dominate subsequent cultures. Healthy cultures should have less than 20% differentiated areas.
4. Enzymatic or non-enzymatic passaging
For enzymatic passaging, Dispase (Catalog #07923) at 1 mg/mL is recommended to passage cells in mTeSR™1 and TeSR™2 cultures. Cells grown in TeSR™2 are slightly more sensitive to dispase than those grown in mTeSR™1, therefore the incubation period should be decreased to 3 - 4 minutes, from 5-7 min for mTeSR™1 cultures (incubation times are based on using dispase from STEMCELL Technologies; may require optimization, as there are differences in enzyme activity from different suppliers). Ensure that dispase is washed off, as residual dispase will continue to cleave the cell surface proteins that are required for the reattachment of cells to the new plates. In addition, reduce the amount of pipetting for TeSR™2 cultures, as TeSR™2 grown clumps break up easier than in mTeSR™1 and can result in undesired single cells that will die upon passage. For more information on enzymatic passaging using dispase, refer to Section 3.2.1. of the Technical Manual.
For non-enzymatic passaging, Gentle Cell Dissociation Reagent (GCDR, Catalog #07174) can be used. For more information using GCDR, refer to Section 3.2.2. of the Technical Manual.
5. Appropriate clump size for passaging
Preparation of a uniform suspension of suitable sized human ES and iPS cell clumps for passaging is critical. Clumps should be approximately 60 um 100 um in diameter (easily determined by an ocular micrometer in the microscope's eyepiece). If the clumps are too large, an increased rate of differentiation within the colonies may occur. If the clumps are too small, with many single cells present, cell survival will be compromised, decreasing the resulting number and health of colonies.
6. Plating density at time of passage
Plating density is a critical aspect of maintaining healthy human ES and iPS cells in mTeSR™1 or TeSR™2. Cultures that are either too sparsely or too densely populated can lead to differentiation. Adjust the plating density and/or split ratios to achieve a balance between having too much space between colonies and having a confluent culture. For the optimal seeding densities for different cultureware, refer to Section 5.3 of the Technical Manual.