Q1:What is the difference between primary cells and cell lines?

A1:According to traditional definition, cells harvested and inoculated for the first time from tissues are called primary cells [Freshney, R.I. (1987). Culture of Animal Cells. A Manual of Basic Technique. (New York, Alan R. Liss, Inc.)]. These cells are directly isolated from tissues, have a limited lifespan, and will gradually stop growing after several passages. To maintain genotypic and phenotypic consistency within the cell population, it is necessary to maximize the growth space during their limited lifespan. Cell lines are continuously growing and differentiating cell populations that have been genetically modified to have unlimited growth potential. In vitro grown cell lines are used for medical or research purposes.

Q2:What is the difference between doubling and passage number?

A2:Doubling refers to the doubling of the total number of cells in culture, usually during the exponential or logarithmic growth phase. Passage number refers to the number of times a cell population is transferred from a culture flask for subculture; the purpose of subculture is to keep cells at low density to stimulate further growth.

Q3:How often should the medium be changed during cell culture?

A3:This depends on the growth rate of the cells. Generally, the medium is changed every 2-3 days. Many cell culture laboratories usually change the medium on Mondays, Wednesdays, and Fridays. Note: After thawing cryopreserved cells, change the medium within 6-16 hours to remove residual DMSO and dead cells.

Q4:Can primary normal human cells be expanded and cryopreserved again?

A4:This depends on the cell type. Some cell types like neurons, glial cells, and some slow-growing epithelial cells are not recommended for expansion and re-cryopreservation. Other cell types like fibroblasts, astrocytes, mesangial cells, etc., can be expanded and cryopreserved again. However, it should be noted that the re-cryopreservation process may cause changes in cell growth performance.

Q5:How much medium should be added to the culture flask?

A5:Our recommended volumes are: T-25 flask: 5ml, T-75 flask: 15ml, T-150 flask: 30ml.

Q6:What to do if primary cells don't adhere well in culture?

A6:Many primary cells require the addition of adhesion-promoting substances to adhere and grow. We can pre-coat the cell culture flasks. Common coating solutions include poly-L-lysine, fibronectin, laminin, vitronectin, gelatin, collagen, MSC adhesion-promoting reagents, Concanavalin A, etc. Alternatively, you can directly purchase consumables with pre-treated surfaces, such as: TC-treated cell culture surfaces; Nunclon Delta cell culture surfaces; flasks for sphere culture, tissue culture, and 3D culture; T25 flasks coated with poly-D-lysine or Type I collagen, etc.

Q7:What to do if cultured cells don't adhere?

A7:Possible reasons:

(1) Over-digestion with trypsin.

(2) Mycoplasma contamination.

(3) Some cells require additional adhesion factors.

(4) Inappropriate culture vessels, such as using non-TC-treated flasks.

Solutions:

(1) Shorten trypsin digestion time or reduce trypsin concentration.

(2) Isolate cultures and test for mycoplasma. Clean the hood and incubator. If mycoplasma contamination is detected, discard the culture.

(3) Change medium composition.

(4) Use appropriate culture vessels.

Q8:What are the precautions for cell thawing and cryopreservation?

A8:Precautions:

(1) When cryopreserving cells, ensure the cells are in good condition: in logarithmic growth phase, at moderate density (usually 70%-80%), and free of contamination;

(2) Handle cells gently during cryopreservation, avoid prolonged digestion, and avoid repeated pipetting;

(3) Use appropriate cell number for cryopreservation: usually 1ml cryopreservation medium per tube, with approximately 2*10⁶ cells. For fragile cells or density-dependent cells, the cell number can be around 5*10⁶;

(4) When cryopreserving cells, ensure the caps are tightly closed and use matching tubes and caps;

(5) Do not place cells directly at -80°C during cryopreservation; strictly perform gradient cooling, and transfer cells from -80°C to liquid nitrogen tank for long-term storage as soon as possible after overnight freezing;

(6) When thawing cells, do not use water bath for too long. If room temperature is low, the water bath temperature can be appropriately increased to 38°C;

(7) After removing from liquid nitrogen tank during thawing, immediately place in water bath to thaw. Gently shake the cryotube to facilitate rapid melting, and centrifuge as soon as possible to remove cryopreservation medium and reduce cell damage;

(8) Pipette cells gently and culture with pre-warmed complete medium.

Q9:What to do if there are many small black dots around and on cells?

A9:Black dots in cells are generally determined to be cell debris or serum precipitate impurities. If suspension cells: collect cell supernatant by low-speed centrifugation (500-600 rpm, 5-6 min) and replace the culture dish; if adherent cells: wash cells 2-3 times with PBS, gently tap the culture dish during washing to detach loosely attached debris and particles, then discard PBS. During digestion, first add low-concentration trypsin (e.g., 0.05%) and digest for about 1 min to detach debris in cell gaps, remove low-concentration trypsin, then digest cells normally. Collect the cell suspension and centrifuge at low speed (500-600 rpm, 5-6 min), replace with a new culture dish, and try increasing serum concentration for culture. If mycoplasma contamination is suspected, perform mycoplasma testing.

Q10:Culture medium appears dirty?

A10:Possible reasons:

(1) Cells just thawed, with many dead cells;

(2) Poor cell condition with increased secretions;

(3) Cell contamination.

Solutions:

(1) Remove all old medium, rinse twice with PBS, and add fresh medium;

(2) Replace with fresh medium and increase serum content;

(3) It is recommended to discard the cells and thaw new ones.

Q11:Severe cell differentiation?

A11:Possible reasons:

(1) Excessive passage number;

(2) Overly vigorous pipetting during passage;

(3) Cells themselves are sensitive.

Solutions:

(1) Reduce passage number and freeze cells at lower passage numbers;

(2) Pipette gently during passage;

(3) For sensitive cells, use gentle reagents and operations, e.g., pre-warm reagents; use trypsin without EDTA; use cell scrapers instead.

Q12:Slow cell growth?

A12: Possible reasons:

(1) Have you changed to a new batch of serum or medium? Cells need time to adapt;

(2) The medium may lack certain growth factors. If the optimal medium is unavailable, cryopreserve some cells under original conditions first, then gradually increase the proportion of new medium: 25%, 50%, 75% to 100%, and let cells adapt after 3-5 passages;

(3) Check if the medium is contaminated. Observe cell condition without antibiotics first;

(4) When seeding density is too low, cell growth slows. Increase seeding density;

(5) Replace with new cells when senescence is observed;

(6) If mycoplasma contamination is suspected, isolate the suspected culture, clean and disinfect the incubator immediately, and use mycoplasma removal reagents or discard the cells.

Q13: Are vacuoles in cells a normal phenomenon?

A13: Some cells naturally contain vacuoles (e.g., HepG2, Huh-7, and some drug-resistant strains), which is normal. If only a few cells have very few vacuoles, it may indicate poor cell condition.

Q14:What to do about vacuolation in cell culture?

A14:Possible reasons:

(1) Cell senescence: Cells are in terminal differentiation or have undergone excessive passages;

(2) pH value: The pH of the medium differs significantly from the optimal pH for cells;

(3) Trypsin digestion: prolonged digestion or excessive pipetting force;

(4) Autophagy: nutrient deprivation induces autophagy;

(5) Cell metabolism: insufficient serum concentration, drug effects, or external stimuli causing metabolic issues;

(6) Contamination: chlamydia, mycoplasma, or viral infection.

Solutions:

(1) If due to senescence, thaw cells with lower passage numbers;

(2) If pH issue, adjust medium to appropriate pH;

(3) Use appropriate trypsin concentration and digestion time, avoid excessive pipetting;

(4) Ensure sufficient nutrients by increasing serum concentration and adding glutamine;

(5) Practice strict aseptic technique and use high-quality FBS from reputable sources.

Q15: What to do if there are many cell debris and dead cells when culturing suspension cells?

A15: Separate cells using stepwise centrifugation at different speeds. First, centrifuge at 200-300 rpm for 3 min to remove dead cell clumps and larger debris, collect the supernatant. Then centrifuge the supernatant at 600-800 rpm for 3 min to remove smaller debris, collect the cell pellet. If there are still many debris, repeat the process multiple times.

Q16:Black particles appear in suspension cell culture

A16:Possible reasons:

(1) Poor cell condition causing aggregation of debris;

(2) Cells grow in large clumps, with internal cells dead due to hypoxia;

(3) Cell contamination.

Solutions:

(1) Low-speed centrifugation to remove debris, increase serum concentration to improve cell condition;

(2) Wash cells with calcium/magnesium-free balanced salt solution, gently pipette to obtain single-cell suspension, seed at low density in plates to gradually remove debris;

(3) Identify the type of contamination and add appropriate antibiotics or mycoplasma removal reagents;

Q17: Semi-adherent/semi-suspension cells don't adhere well and easily detach?

A17: Handle cells carefully, avoid shaking plates vigorously. When changing medium, pre-warm medium to 37°C and slowly dispense against the wall to minimize cell disturbance. Some cells require coating for adhesion. Common coating solutions include: collagen, polylysine, fibronectin, gelatin, etc.

Q18: Solutions for cell detachment and rolling during handling

A18: Poorly adherent cells are prone to detachment when stimulated at high density. Handle such cells by pre-warming media/reagents to avoid sudden temperature changes; use pipette to gently dispense reagents against container walls; if detachment occurs, trypsinize, resuspend, and re-seed cells.

Q19:What causes cell detachment in culture?

A19: Causes of cell detachment in culture:

(1) Is the pH of the medium appropriate (usually pH 7.3-7.4)? During culture, active metabolism lowers pH and medium turns yellow (with phenol red indicator). Change medium promptly.

(2) Cell density - some cells cannot grow too dense and require timely passage;

(3) If detachment just begins, change medium immediately to wash away dead cells, as substances from dead cells can trigger massive apoptosis.

Q20: Why do cells round up when digested with trypsin?

A20: Trypsin digestion causes adherent cells to round up because after trypsin treatment, cells lose their protective layer and growth ability weakens. Under the tension of internal cytoskeleton, cells become spherical. Prolonged trypsin exposure also digests cell membrane proteins, damaging cells. Use with caution.

Q21: How to resolve cell aggregation in experiments?

A21: When culturing suspension cells, cells may aggregate into clumps for various reasons.

The most common cause is free DNA and cell debris in the medium, usually after cell lysis. The sticky nature of DNA causes cells and debris to aggregate into large clumps.

Here are some causes of cell lysis and DNA release into the medium:

(1) Over-digestion: Excessive treatment with proteolytic enzymes like trypsin causes cell clumping;

(2) Environmental stress: Mechanical force, repeated freeze-thaw, and other stresses accelerate cell death, with cell adhesion as an early symptom;

(3) Tissue dissociation: Preparing single-cell suspensions from primary tissue via chemical, mechanical, or enzymatic methods may rupture some cells. Collagenase digestion is typically used to break down extracellular matrix;

(4) Overgrowth: When cells reach confluence, cell debris and free DNA accumulate excessively.

Q22:How to determine if cells are properly digested?

A22:First, understand: Proper digestion is not defined as complete single-cell dispersion. Visually, when the adherent cell layer starts to detach and flows like sand grains, cells have detached from the substrate and cell-cell connections are broken. Cells are independent but not fully dispersed - stop digestion immediately.

Second, note: Do not wait until all cells are completely separated with large gaps under the microscope. Even as a single-cell suspension, cells will reaggregate during attachment - this is inherent to adherent cells (especially tumor cells).

Finally, consider: Abnormal cell morphology may indicate improper digestion. However, if digestion is correct but cells still form sheets (even after thorough pipetting), this reflects cell characteristics - they reaggregate during attachment. Forcing uniform distribution damages cell condition.

Q23: How to perform complete medium change?

A23: For adherent cells, use complete medium change: aspirate all old medium and add sufficient fresh complete medium.

Complete medium change procedure:

(1) Place equipment in laminar flow hood, turn on UV sterilization. For temperature-sensitive cells, warm serum-containing medium to 37°C in water bath before transferring to hood. UV-irradiate culture room for ~30 minutes.

(2) Remove flask from incubator, spray surface with 75% ethanol, transfer to hood. Open cap, gently aspirate old medium. Add fresh serum-containing medium using pipette - avoid adding directly onto cell layer; dispense against the wall gently to prevent detachment.

(3) Cap flask, label with medium change time, cell type, and operator information.

(4) Before returning to incubator, spray flask surface with ethanol again. Clean hood surface with ethanol and dispose of waste.

Q24: How to perform partial medium change?

A24: "Partial medium change" means discarding half the old medium and adding half fresh medium. Reasons for partial change:

(1) Provides buffered environment for adaptation;

(2) Cells may secrete growth factors during growth;

(3) Saves materials;

(4) Convenience: e.g., 96-well plates are difficult to change completely. Multichannel pipettes are commonly used for partial changes;

(5) For suspension cells, partial change reduces cell density;

Q25: How to change medium for suspension cells?

A25: Procedure:

(1) Aspirate half the old medium;

(2) Add fresh complete medium (half the cells will be lost).

To avoid losing cells, transfer culture to centrifuge tube, centrifuge to remove old medium, then resuspend pellet in fresh medium.

Q26: Is PBS wash necessary during medium change?

A26: We know that during "passaging adherent cells", serum in the medium inhibits trypsin activity and affects digestion, so PBS wash is essential.

However, for medium change, no clear guidelines exist regarding PBS wash.

Consider cell-specific conditions:

a.For delicate cells or poor growth conditions, avoid PBS wash. PBS rinsing may disrupt weak cell adhesion and wash away secreted growth factors. If washing is necessary, do so gently;

b.For healthy cells or when medium appears dirty, PBS wash can remove accumulated metabolites and wash away poorly growing/senescent cells to maintain viability.

Q27: What precautions should be taken during medium change?

A27: Precautions:

(1) Maintain sterility throughout: wear sterile gown, gloves, and mask. Handle flask openings carefully to avoid contamination;

(2) Do not add medium directly onto adherent cells;

(3) Observe and photograph cell condition before changing medium.

Q28: What is the effect of serum quality on cell culture?

A28: Serum is a key component of cell culture medium. Poor quality serum may contain toxic substances, microbial contaminants, or have low growth factor content, which can affect cell growth and experiment results.

Q29: How to preserve cell culture medium?

A29: Store medium at 2-8°C in the dark. Avoid repeated freeze-thaw cycles. After preparation, filter sterilize and store in aliquots. Complete medium containing serum should be used within 2-4 weeks.

Q30: What is the role of antibiotics in cell culture?

A30: Antibiotics are used to prevent bacterial contamination in cell culture. Common antibiotics include penicillin, streptomycin, and amphotericin B. However, long-term use of antibiotics may mask contamination and affect cell metabolism.

Q31: How to troubleshoot common cell culture problems?

A31: Troubleshooting steps: (1) Check culture environment: temperature, CO2 concentration, humidity; (2) Examine medium: pH, serum quality, nutrient components; (3) Inspect cells: morphology, density, contamination; (4) Review experimental operations: aseptic technique, reagent handling, incubation conditions. Systematic elimination helps identify root causes.