Primary cell culture involves the isolation and growth of cells directly from tissues cultured in vitro for experimental research. Primary cells reflect the biological characteristics of the tissue they are derived from, making them a valuable tool in biological research, drug testing, and disease modeling. Below is a detailed protocol for setting up primary cell cultures.
Primary Cell Culture Protocol
Materials Needed
1. Tissue Source: Organ or tissue from which cells are to be isolated (e.g., liver, lung, skin, etc.).
2. Enzymes for Tissue Dissociation
Collagenase (typically used for soft tissues).
Trypsin (to detach cells from tissues or culture plates).
DNase (to break down DNA released from dead cells).
Dispase or hyaluronidase (for tougher tissues).
3. Culture Media
Choose a culture medium appropriate for the cell type (e.g., DMEM, RPMI-1640, or specialized media such as Keratinocyte Serum-Free Media (KSFM) or Endothelial Cell Growth Media).
Supplements like FBS (Fetal Bovine Serum), growth factors (EGF, bFGF), antibiotics (penicillin-streptomycin), and glutamine.
- Cell Strainers (40-70 µm) to filter cell suspension.
- Sterile Petri Dishes, culture plates, or flasks (depending on the intended culture format).
- Sterile PBS (phosphate-buffered saline).
- Trypsin-EDTA or enzyme-free dissociation buffer (if necessary).
- Centrifuge.
- Laminar Flow Hood and Incubator: Standard tissue culture equipment to maintain sterility and temperature (typically 37°C with 5% CO₂ for mammalian cells).
Procedure
Preparation and Sterilization
Sterilize all tools: Dissecting instruments (scalpels, scissors, forceps), Petri dishes, and pipettes should be sterilized by autoclaving or using ethanol.
Work in a sterile environment: All procedures should be conducted under a laminar flow hood to maintain sterility.
Prepare the culture medium: Warm the appropriate medium supplemented with FBS, antibiotics, and any necessary growth factors to 37°C in advance.
Tissue Collection and Dissection
Collect the tissue: Immediately after excision, place the tissue in a sterile container with cold PBS or transport media (such as HBSS or DMEM) to keep it viable during transfer.
If obtaining tissue from an animal, ensure proper ethical approvals and protocols for handling the tissue.
Dissect the tissue: Under sterile conditions in the hood, cut the tissue into small pieces (~1–2 mm³) using sterile scalpels or scissors. The finer the pieces, the more efficient the digestion process will be.
Tissue Digestion and Cell Isolation
Enzymatic Digestion: To dissociate cells from the tissue matrix:
Transfer the minced tissue into a sterile conical tube.
Add digestion enzymes (e.g., collagenase, trypsin) prepared in warm PBS or culture medium.
Example: For collagenase digestion, use 1-2 mg/mL of collagenase in PBS.
Incubate the tissue with the enzyme solution at 37°C in a shaking water bath or incubator for 30 minutes to 2 hours, depending on the tissue type.
Agitate the tube occasionally to promote dissociation.
After incubation, gently pipette the solution up and down to help further dissociate the tissue into single cells.
Filtration and Centrifugation
Filtration: Pass the cell suspension through a sterile cell strainer (40–70 µm) into a new sterile tube to remove undigested tissue fragments.
Centrifugation: Centrifuge the filtered cell suspension at 300–400 x g for 5-10 minutes at room temperature.
This step will pellet the cells, allowing you to discard the supernatant containing the digestion enzymes and other debris.
Resuspend the Pellet: After centrifugation, carefully aspirate the supernatant and resuspend the cell pellet in fresh culture medium.
Seeding and Culturing Cells
Cell Counting (Optional): At this point, count the cells using a hemocytometer and trypan blue staining to assess cell viability and ensure accurate seeding density.
Seeding: Seed the cells into sterile culture flasks, dishes, or plates at an appropriate density (e.g., 5 x 10⁴ to 1 x 10⁶ cells depending on the cell type and experiment).
Use specialized-coated plates (e.g., collagen, fibronectin, or gelatin-coated plates) for certain cell types, such as endothelial or epithelial cells, which require adherence support.
Incubation: Place the culture flasks or plates in a 37°C incubator with 5% CO₂. Ensure proper culture conditions specific to the cell type being used (e.g., low oxygen for certain primary cell types).
Monitoring Cell Growth
Observe Cells: Over the next 24-48 hours, check the cells regularly under a microscope to monitor attachment, spreading, and growth.
Primary cells typically grow slower than immortalized cell lines, so they may take a few days to adhere and proliferate.
Medium Change: Replace the culture medium every 2-3 days, carefully aspirating the old medium and replacing it with fresh, pre-warmed medium.
Ensure the cells remain in a healthy monolayer and are not over-confluent (typically 70-90% confluency is ideal for subculturing).
Subculturing (Passaging)
Subculturing: When the cells reach appropriate confluence (~70-90%), they should be passaged to avoid overgrowth and maintain their health.
Detachment: Gently wash the cells with sterile PBS to remove any residual medium.
Add trypsin-EDTA or a gentle dissociation buffer to detach the cells. Incubate at 37°C for 2-5 minutes, monitoring under the microscope until the cells round up and begin detaching.
Neutralization: Neutralize the trypsin with fresh complete medium containing serum (FBS). Pipette up and down to create a single-cell suspension.
Centrifuge the cell suspension at 300–400 x g for 5 minutes.
Reseeding: Resuspend the cells in fresh medium and reseed them into new culture dishes at a lower density (e.g., 1:3 or 1:5 split ratios, depending on the cell type).
Cryopreservation (Optional)
If you wish to store primary cells for future use, you can cryopreserve them:
Freezing Medium: Prepare freezing medium (10% DMSO in FBS or complete culture medium).
Cryovials: Aliquot 1–2 million cells per cryovial in freezing medium.
Freezing: Gradually freeze the cells by placing them in a controlled-rate freezing container (-80°C freezer) overnight before transferring them to liquid nitrogen for long-term storage.
Critical Tips and Considerations
Tissue-Specific Conditions: Each tissue type has specific requirements for dissociation enzymes, media, and growth conditions. Consult protocols or literature specific to your cell type of interest.
Sterility: Maintaining sterility throughout the procedure is critical to avoid contamination.
Cell Type-Specific Media: Primary cells often require specialized growth media with specific supplements like growth factors (e.g., EGF, insulin, hydrocortisone).
Lifespan: Primary cells have a limited lifespan in culture, meaning they can only be passaged a finite number of times before undergoing senescence. Avoid too many passages to maintain the biological relevance of the cells.
Conclusion
Primary cell culture is a valuable tool for studying physiological processes in a more relevant context compared to immortalized cell lines. Following the steps in this protocol will allow for the successful isolation, culture, and maintenance of primary cells, although specific modifications may be needed depending on the tissue type and cell characteristics. Proper handling, growth conditions, and regular monitoring are essential for preserving the health and viability of primary cells.