Tips and tricks for iPSC culture

Top tips for successful induced pluripotent stem cell and embryonic stem cell culture.

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In the field of regenerative medicine and developmental biology research, the basic technique of culturing pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), is crucial. Nevertheless, keeping PSCs from differentiating can be quite problematic. This article will offer some nuggets of wisdom to aid in the successful and consistent culture of PSCs.

1. Start with High-Quality Cell Culture Vessels and Extracellular Matrices (ECM)

Invest in high-quality cell culture dishes or plates specifically designed for PSCs. Most PSC cultures require extracellular matrices (ECM). Select culture dishes that are compatible with the coating of ECMs like Matrigel, Geltrex, Laminin, fibronectin, etc.

Choose ECMs that are animal component-free to ensure better consistency in the results as well as avoid any contaminants that can potentially come from animal sources.

2. Regularly Inspect and Clean Equipment

To ensure the viability and expansion of your iPSC cultures, maintaining a sterile environment is crucial. Guard against contamination by thoroughly examining and sanitizing key equipment like incubators, hoods, and pipettes. Contaminants, such as bacteria, fungi, or mycoplasma, can jeopardize the integrity of your iPSC cultures. Traces of such impurities, no matter how tiny, may undermine the accuracy of your results or render your research unviable. Properly employing disinfectants and following strict routines is the only way to maintain aseptic conditions.

3. Optimize Media Formulations

Select a defined and reliable PSC culture medium that supports pluripotency. Media should be supplemented with essential components such as growth factors, serum replacement, and small molecules. Growth factors play a major role in maintaining the pluripotency. Choose growth factors derived from optimal expression systems. For example, growth factors derived from a Human expression system are superior to a bacterial system as they have enhanced bioactivity and stability in the cell culture media. Try using animal component-free growth factors to yield better experimental consistency. Ensure that the media and its components are not used post the expiry date.

Stability of HumanKine thermostable bFGF-TS in xeno free media w/o cells

Stability of HumanKine bFGF-TS in iPSC culture compared to normal bFGF

By: Nick Asbrock, Christine Chen and Vi Chu*, EMD Millipore, Bioscience Division, Temecula, CA, USA.

4. Handle Cells Gently

iPSCs are highly sensitive to mechanical stress. Vigorous pipetting or harsh detachment methods, can damage cell membranes, disrupt cell-cell interactions, and lead to a loss of cell viability. When pipetting iPSCs, use wide-bore pipette tips to minimize shear force on the cells. These tips reduce the chances of damaging cells during aspiration and dispensing. When passaging iPSCs, choose a gentle detachment method using EDTA or dispase.

5. Regularly Monitor Cell Density

One of the most important factors in successful PSC cultures is optimal cell density. Too few cells may not be able to absorb enough nutrients, while too many cells can lead to nutrient loss and waste accumulation. Maintaining proper cell density is essential for maintaining their pluripotency. Too many cells can cause differentiation, resulting in lost pluripotency, and unwanted cell types.

6. Use ROCK Inhibitor During Passaging

Using a Rho kinase (ROCK) inhibitor during passaging is a valuable technique, especially when working with sensitive cell types like induced pluripotent stem cells (iPSCs). iPSCs can be vulnerable to stress during passaging, which can lead to cell death. ROCK inhibitors mitigate cell stress, improving cell survival rates during detachment, reseeding, and attachment.

7. Regularly Test for Mycoplasma Contamination

Mycoplasma contamination can often go unnoticed and affect the survivability and pluripotency of PSC cultures. Mycoplasma can negatively impact the differentiation experiments leading to the wastage of valuable reagents and samples. Although there are recommended protocols to clear mycoplasma in cell cultures using some antibiotics, studies have shown mixed results. If you detect mycoplasma in your PSC cultures, it is better to get rid of the infected sample and start fresh.

9. Check Pluripotency Before Starting Differentiations.

Pluripotency is the ability of a cell to differentiate into any of the three cell types in the three layers of the germ: endoderm, mesoderm, or ectoderm. It is important to ensure that your iPSC(s) or ESC(s) are truly pluripotent before differentiation to obtain accurate and meaningful results in subsequent experiments. Pluripotency can be measured by employing specific antibodies to detect pluripotency markers in Immunocytochemistry, flow cytometry, or genetic analysis using qPCR, etc. It is critical to start with at least a 95% pluripotent stem cell population for any differentiations.

iPSC differentiated to definitive endoderm using HumanKine Animal component free growth factors and characterized using Proteintech antibodies.

9. Maintain Records and Documentation

It is essential to maintain a comprehensive record of your culture conditions and passaging protocols, as well as any observed cell behavior. This documentation is of great value for the resolution of issues and the purpose of reproducibility. Additionally, it is beneficial to stay abreast of the most recent developments in PSC culture methods and technologies. By networking and collaborating with researchers from the field, one can gain valuable knowledge and solutions to common issues.

Conclusion

Culturing pluripotent stem cells is both an art and a science. By following these tips and tricks and maintaining strict adherence to best practices, you can enhance the success and reproducibility of your PSC cultures. Whether you're studying developmental biology, regenerative medicine, or disease modeling, mastering PSC cultures is a critical step toward advancing your research and unlocking the full potential of pluripotent stem cells.

Proteintech reagents for advancing pluripotent stem cell research

Antibodies

Pluripotency marker antibodies
	Marker	Proteintech Antibodies	Number of citations
	Nanog	14295-1-AP	184
	OCT4	11263-1-AP	197
	SOX2	11064-1-AP	212
	KLF4	11880-1-AP	106
	Endoderm marker antibodies
	Marker	Proteintech Antibodies	Number of citations
	SOX17	24903-1-AP	7
	FOXA2	22474-1-AP	16
	CXCR4	60042-1-Ig	36
	Mesoderm marker antibodies
	Marker	Proteintech Antibodies	Number of citations
	NCAM1	14255-1-AP	41
	NKX 2.5	13921-1-AP	12
	TBX6	12447-1-AP
	Ectoderm marker antibodies
	Marker	Proteintech Antibodies	Number of citations
	Nestin	19483-1-AP	70
	PAX6	12323-1-AP	49
	OTX2	13497-1-AP	22

HumanKine Growth factors

Pluripotent stem cell(PSC) maintenance
Growth factor	HumanKine	Number of citations
bFGF	HZ-1285	50
TGFb1	HZ-1011	156
	Growth factors for germ layer differentiations
Growth factor	Proteintech products	Number of citations
Activin A	HZ-1138	25
Noggin	HZ-1118	13
BMP2	HZ-1128	32
BMP4	HZ-1045	69
bFGF	HZ-1285	50
	Growth factors for Neural Differentiation
Growth factor	Proteintech products	Number of citations
bFGF	HZ-1285	50
EGF	HZ-1326	10
beta NGF	HZ-1222
BDNF	HZ-1335
GDNF	HZ-1311	1
FGF8B	HZ-1103	3
CNTF	HZ-1331
DKK1	HZ-1314
FGF19	HZ-1330	1

	Growth factors for Cardiac differentiation
Growth factor	Proteintech products	Number of citations
Activin A	HZ-1138	25
BMP4	HZ-1045	69
bFGF	HZ-1285	50
VEGF165	HZ-1038	29
VEGF121	HZ-1204	18
	Growth factors for Pancreatic differentiation
Growth factor	Proteintech products	Number of citations
HGF	HZ-1084	14
BMP4	HZ-1045	69
IGF1	HZ-1322
Activin A	HZ-1138	25
	Growth factors for Hepatocyte differentiation
Growth factor	Proteintech products	Number of citations
bFGF	HZ-1285	50
Wnt3A	HZ-1296	2
HGF	HZ-1084	14
OSM	HZ-1030	5
FGF19	HZ-1330
Rspondin 1	HZ-1328	1
BMP4	HZ-1045	69
EGF	HZ-1326	10