Can I use the Poly-D-Lysine matrix with other coatings?
iPSC-derived neurons have been cultured successfully on Poly-D-Lysine with a secondary coating of mouse and human laminin.
I am interested in using StemFlex Medium in somatic cell reprogramming. What protocol should I follow for blood cells?
We have evaluated the use of Cytotune-iPS 2.0 Sendai Reprogramming Kit (Cat. No. A16517) for somatic cell reprogramming of CD34+ blood cells. For CD34+ cells, follow the instructions provided in the Cytotune 2.0 reprogramming manual for feeder-free reprogramming (pgs. 39-44). On Day 3, you can utilize rhVTN-N (Cat. No. A14700), Geltrex (Cat. No. A1413302), or rhLaminin-521 (Cat. No. A29248 or A29249). From Day 8 onward, rather than feeding daily with Essential 8 Medium, reprogrammed CD34+ cells should be fed every-other-day with StemFlex Medium.
I am interested in using StemFlex Medium in somatic cell reprogramming. What protocol should I follow for human fibroblasts?
We have evaluated the use of Cytotune-iPS 2.0 Sendai Reprogramming Kit (Cat. No. A16517) for somatic cell reprogramming of both neonatal and adult human dermal fibroblasts. For fibroblasts, follow the instructions provided in the Cytotune 2.0 reprogramming manual for feeder-free reprogramming (pgs. 16-20). On Day 7, you may use rhVTN-N (Cat. No. A14700), Geltrex matrix (Cat. No. A1413302), or rhLaminin-521 (Cat. No. A29248 or A29249). From Day 8 onward, rather than feeding daily with Essential 8 Medium, we recommend that you feed reprogrammed fibroblasts every-other-day with StemFlex Medium.
Can I use Geltrex matrix instead of rhLaminin-521 for clonal expansion workflows where cells are seeded at 1-5 cells per well of a 96-well plate?
We have not yet evaluated the Geltrex matrix system for clonal expansion in the presence of StemFlex Medium. However, rhLaminin-521 does provide optimal survival of cells following single-cell passaging and thus this matrix is recommended for such critical applications.
What protocol is recommended for delivery of Cas9 protein:guide RNA complex to my PSCs cultured in StemFlex Medium?
We recommend use of the Neon Electroporation device for electroporation of PSCs with Cas9 protein:guide RNA complex following the protocol guidance in the following demonstrated protocol (https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/PG1584-PJT1313-COL31227-Demonstrated-Protocol-Performing-CRISPR-Cas9-FHR.pdf); see the section entitled “Knockout by electroporation of RNP using the Neon Transfection System.” We have seen that the Neon electroporation protocols 7 and 14 provide optimal indel formation while maintaining cell survival. However, the electroporation conditions may need to be optimized for your pluripotent cell line.
You recommend Geltrex as the matrix for routine culture of StemFlex Medium. Can I also use Vitronectin or rhLaminin-521?
Yes. We recommend following the coating instructions for Vitronectin or rhLaminin-521 and using the culture and passaging recommendations in the StemFlex protocol.
Do I need to use ROCK inhibitor during single-cell passaging in StemFlex Medium?
The use of ROCK inhibitor is not required when culturing in StemFlex Medium on rhLaminin-521. However, supplementation with RevitaCell Supplement can provide additional support to PSCs during stressful transitions such as single-cell passaging.
Can pluripotent stem cells (PSCs) cryopreserved in a different culture condition be thawed and subsequently cultured in Gibco Essential 8 Medium and on vitronectin (VTN-N)?
Yes. PSCs cryopreserved from cultures of mTeSR Medium and BD Matrigel Basement Membrane Matrix may be thawed into Gibco Essential 8 Medium and plated on VTN-N. Certain lines may benefit from thawing into the medium and substrate they were growing in at the time of cryopreservation. Then at the next passage, use EDTA to passage the cells into Gibco Essential 8 Medium and VTN-N.
What cell lines have been successfully tested with Gibco Essential 8 Medium under feeder-free conditions?
There have been multiple pluripotent stem cell (PSC) lines tested with the Gibco Essential 8 Medium System (https://www.ncbi.nlm.nih.gov/pubmed/?term=21478862).
What is the role of bFGF in Gibco Essential 8 Medium?
bFGF is vital for pluripotent cell survival and proliferation.
Can Gibco RevitaCell Supplement be used with any PSC growth medium?
We have tested and confirmed utility with the following PSC growth media: Gibco Essential 8 Medium, Gibco StemPro hESC SFM, mTesR1 medium, and Gibco KnockOut Serum Replacement (KSR)- containing feeder-dependent medium.
Can fibroblasts obtained from skin biopsy samples be cultured in Gibco Essential 8 Medium to achieve xeno-free conditions?
Yes, fibroblasts from skin biopsy samples can be expanded and cultured in Gibco Essential 8 Medium with the addition of EGF, thrombin, and hydrocortisone (http://www.ncbi.nlm.nih.gov/pubmed/21478862).
What is the role of insulin in Gibco Essential 8 Medium?
Insulin is important for cell survival and proliferation.
Can human PSCs be frozen in Gibco Essential 8 Medium?
Yes, cells can be routinely frozen in complete Gibco Essential 8 Medium and 10% DMSO. Alternatively, ready-to-use CTS PSC Cryomedium (Cat. No. A4238801) provides more efficient post-thaw recovery.
Can I use enzymes such as dispase and collagenase for passaging cells cultured in Gibco Essential 8 Medium and on VTN-N?
Enzymes such as dispase and collagenase do not work well with cells cultured in Gibco Essential 8 Medium on VTN-N. Use of these enzymes for passaging cells results in compromised viability and attachment.
What is the recommended passaging method to use with Gibco Essential 8 Medium and on Gibco Vitronectin (VTN-N)?
Cells cultured in Gibco Essential 8 Medium and VTN-N need to be passaged with EDTA.
Can I feed the cells grown in Gibco Essential 8 Medium less frequently than those in other feeder-free medium?
No. The cells should be fed daily including the day after passaging.
Can Rho-associated protein kinase (ROCK) inhibitors be used in Gibco Essential 8 Medium?
Yes, ROCK inhibitors can be used with Essential 8 Medium. We suggest using RevitaCell Supplement (Cat. No. A2644501), which has been specifically designed to minimize the impact of stress on PSCs.
Can I warm Gibco Essential 8 Medium in a 37 degrees C waterbath for daily use?
It is very important that complete Gibco Essential 8 Medium is prewarmed at room temperature and not in a 37 degrees C water bath. bFGF activity can decline rapidly with repeated temperature changes from 4 degrees C to 37 degrees C.
What is the shelf life of complete Gibco Essential 8 Medium?
The shelf life of complete Gibco Essential 8 Medium is two weeks at 2-8 degrees C.
Can I thaw the frozen Gibco Essential 8 Supplement (50X) in a 37 degrees C waterbath?
We recommend thawing the supplement at room temperature for approximately 1 hour.
Can I use other versions of DMEM/F-12 in place of the Gibco Essential 8 Basal Medium supplied with the kit?
Other catalog versions of DMEM/F-12 cannot be used in place of the Gibco Essential 8 Basal Medium in the preparation of Gibco Essential 8 Medium. Gibco Essential 8 Basal Medium supplied with the kit has a higher level of sodium bicarbonate.
How do I prepare Gibco Essential 8 Medium?
To prepare 500 mL of complete Gibco Essential 8 Medium, thaw Gibco Essential 8 Supplement (50X) at room temperature for 1 hour and then aseptically combine the components listed below:
- Gibco Essential 8 Basal Medium: 490 mL
- Gibco Essential 8 Supplement (50X): 10 mL
What will my cells look like growing in Gibco Essential 8 Medium and on Gibco Vitronectin (VTN-N)?
You should expect to see normal pluripotent stem cell (PSC) morphology. The expected morphology of PSCs is demonstrated specifically by tightly packed colonies with defined borders and a high nucleus-to-cytoplasm ratio.
Can pluripotent stem cells (PSCs) previously cultured in other media and on other substrates be cultured in Gibco Essential 8 Medium and on Gibco Vitronectin?
Cells cultured in other feeder-free media systems, such as mTeSR Medium with Matrigel Basement Membrane Matrix, or StemPro hESC SFM with Geltrex Matrix, can be successfully cultured in Gibco Essential 8 Medium and VTN-N. In addition, PSCs grown on feeders with KnockOut SR have also been shown to be successfully cultured in Gibco Essential 8 Medium on VTN-N. However, when changing media systems, cells must be passaged either manually, or with EDTA prior to culturing in Gibco Essential 8 Medium on VTN-N.
Does Gibco Essential 8 Medium exhibit variability from lot to lot?
Gibco Essential 8 Medium has reduced variability compared to existing feeder-free culture media. Unlike other media that contain over 20 highly variable ingredients, Gibco Essential 8 Medium is produced under cGMP and has an optimized formulation and growth factor levels that help ensure maximum cell health, pluripotency, and growth, with minimal variability.
Can Gibco Essential 8 Medium and Gibco Vitronectin (VTN-N) support long-term growth of pluripotent stem cells (PSCs)?
Gibco Essential 8 Medium and vitronectin have been shown to support PSC growth for >50 passages without any signs of karyotypic abnormalities, and maintain the ability of PSCs to differentiate into all three germ line lineages. As published by Chen et al (http://www.ncbi.nlm.nih.gov/pubmed/21478862) in the laboratory of James Thomson, the VTN-N variant of vitronectin supports human pluripotent stem cell attachment and survival better than wild-type vitronectin when used in conjunction with Gibco Essential 8 Medium.
Is Gibco Essential 8 Medium xeno-free (human or recombinant origin only)?
Yes. Gibco Essential 8 Medium provides reliable and robust cultures with a xeno-free, eight-component medium
What are the main differences to be taken into consideration when culturing cells in Gibco Essential 8 Medium on Gibco Vitronectin (VTN-N) compared to other feeder-free systems?
Here are three major differences to be taken into consideration when culturing cells in Gibco Essential 8 Medium on Gibco Vitronectin (VTN-N) compared to other feeder-free systems:
- Cells should be typically passaged ~24 hours sooner than they would be with other feeder-free media.
- Passaging should take place when cells are at ~85% confluency. If cells are passaged when they are more than 85% confluent, the health of the cells and final cell yield may be compromised.
- Cells must be passaged in EDTA. Collagenase and dispase are not recommended.
Does Gibco Essential 8 Medium already contain bFGF?
Yes. Gibco Essential 8 Medium contains 100 ng/mL basic fibroblast growth factor (bFGF), and no additional bFGF is required.
How many components are there in Gibco Essential 8 Medium?
Gibco Essential 8 Medium contains only the eight required components for culturing PSCs. The medium was developed by Chen et al (http://www.ncbi.nlm.nih.gov/pubmed/21478862) to overcome the variability issues observed with mTeSR Medium. Gibco Essential 8 Medium is designed to have less variability due to limited components and removal of albumin (BSA) from the formulation. Gibco Essential 8 Medium is provided as a convenient two-component kit: 500 mL Gibco Essential 8 Basal Medium and 10 mL Gibco Essential 8 Supplement (50X).
What is Gibco Essential 8 Medium?
Gibco Essential 8 Medium is a xeno-free and feeder-free medium specially formulated for the growth and expansion of human pluripotent stem cells (PSCs). Originally developed by Chen et al (http://www.ncbi.nlm.nih.gov/pubmed/21478862) in the laboratory of James Thomson, and validated by Cellular Dynamics International, Gibco Essential 8 Medium has been extensively tested and is proven to maintain pluripotency in multiple PSC lines.
Can I use EDTA or versene for passaging of stem cells?
Yes, you may use EDTA or versene. However, we do not recommend using dispase or collagenase as it can lead to differentiation.
Does the recombinant human laminin-521 (rhLaminin-521) have to be on ice before use?
We recommend that you leave the product on ice while prepping to use it.
Do recombinant human laminin-521 (rhLaminin-521) pre-coated plates have to be covered with liquid at all times?
Yes, do not let the laminin dry out on the plates.
Can PSCs transitioned over into Essential 8 Medium on recombinant human laminin-521 (rhLaminin-521) from feeder-dependent cultures, then be transferred onto vitronectin (VTN-N)?
Yes. Following 2 passages on the rhLaminin-521 matrix, Versene or EDTA passaging should be used to subculture PSCs.
Can recombinant human laminin-521 (rhLaminin-521) be used at coating concentrations lower than 0.5 ug/cm2?
The optimal working concentration of rhLaminin-521 is cell line dependent and must be determined empirically. However, for some cell lines, coating concentrations as low as 0.3 µg/cm2 can be used with no decrease in performance. Additionally, coating plates overnight at 4 degrees C can support coating concentrations as low as 0.1 µg/cm2.
Can recombinant human laminin-521 (rhLaminin-521) be used with Essential 8 Flex Medium?
Yes. To ensure optimum recovery of PSCs following single-cell passaging, PSCs should be fed with Essential 8 Flex Medium the day before passaging.
