Cell isolation and preparation are fundamental steps in various biological and biomedical research areas, including virology, immunology, and cell biology. Obtaining cells for cell culture typically involves isolating cells from animal or human tissues, or using established cell lines. The choice of source depends on the type of experiment, the desired cell type, and the goals of the research.
Where and how do we obtain the cells?
1. Objectives of Cell Isolation
- Obtain pure populations: For studying specific cell types or processes.
- Maintain viability: Ensure cells are functional for downstream applications.
- Minimize contamination: Avoid cross-contamination with other cell types or organisms.
2. Sample Sources
- Blood: White blood cells, red blood cells, platelets.
- Tissues: Liver, spleen, lungs, etc., for extracting primary cells.
- Cell Cultures: Adherent or suspension cells.
3. General Workflow
Step 1: Tissue Dissociation
- For solid tissues:
- Mechanical disruption: Using homogenizers or sieves.
- Enzymatic digestion: Collagenase, trypsin, or dispase to break down extracellular matrix.
- Chemical dissociation: EDTA or other chelators to disrupt cell adhesion.
Step 2: Centrifugation and Filtration
- Use gradient or differential centrifugation to separate cells based on density or size.
- Filter cell suspensions to remove debris and clumps.
Step 3: Purification Techniques
- Density gradient centrifugation: Ficoll or Percoll to separate cells based on buoyant density.
- Magnetic-Activated Cell Sorting (MACS): Uses magnetic beads coated with specific antibodies.
- Fluorescence-Activated Cell Sorting (FACS): Uses flow cytometry for high-purity isolation of cells labeled with fluorescent markers.
4. Specific Protocols
a) Isolation from Primary Tissues
Primary cells are directly isolated from living tissues.
- Sources
- Animal tissues (e.g., liver, kidney, lungs, or skin from mice, rats, or larger mammals).
- Human tissues (e.g., biopsies, surgical specimens, or donated tissues).
- Procedure
- Dissection and Enzymatic Digestion
- Tissues are minced and treated with enzymes (e.g., trypsin, collagenase, or dispase) to separate cells.
- Mechanical Disruption
- Physical methods (e.g., homogenization or grinding) are used to break tissues into smaller fragments.
- Filtration
- The suspension is filtered to remove debris, leaving a cell-rich solution.
- Centrifugation
- Used to collect and wash cells before plating them in culture media.
- Dissection and Enzymatic Digestion
- Challenges
- Primary cells are often difficult to culture and have a limited lifespan in vitro.
- a. The tumor tissue should be obtained immediately after surgery, surrounding tumor tissue is removed. Bath for 10 min in PBS + 10% antibiotics
- b. cut into very small pieces (1–2 mm3) by scissors or scalpels in PBS or media
- c. After disaggregation of the tumor tissue into small pieces
- d. Centrifuge the suspension collected in PBS or media
- The different fractions should be set in suitable media
b) Established Cell Lines
These are immortalized cells that can be cultured indefinitely.
- Sources
- Public cell repositories (e.g., ATCC, ECACC, or DSMZ).
- Laboratories sharing their established cell lines.
- Examples
- HeLa cells: Cervical cancer cells from humans.
- MDCK cells: Canine kidney epithelial cells.
- Vero cells: Monkey kidney cells.
- Advantages
- Easy to culture and widely characterized.
- Reliable and reproducible for experiments.
- Challenges
- Genetic or phenotypic drift over time if not carefully maintained.
c) Stem Cells
Stem cells can differentiate into various cell types and are increasingly used for specific studies.
- Sources
- Embryonic stem cells: Derived from early embryos.
- Adult stem cells: Harvested from tissues like bone marrow or fat.
- Induced pluripotent stem cells (iPSCs): Adult cells reprogrammed to a pluripotent state.
- Procedure
- Isolation, expansion in defined media, and differentiation into specific cell types.
- Applications
- Regenerative medicine and developmental studies.
d) Peripheral Blood or Bone Marrow
Some cells, like lymphocytes, are derived from blood or bone marrow.
- Procedure
- Blood is collected and subjected to density gradient centrifugation (e.g., using Ficoll-Paque) to isolate mononuclear cells.
- Cultured in appropriate conditions (e.g., mitogen stimulation for lymphocyte activation).
e) Organotypic and Explant Cultures
Small pieces of tissue are maintained in culture to preserve their architecture.
- Applications
- Studying tissue-specific responses or drug effects.
Key Considerations in Cell Culture Preparation
- Aseptic Technique: Essential to prevent contamination.
- Media and Supplements: Cells need specific nutrients, growth factors, and gases (e.g., CO₂) to thrive.
- Ethics and Permissions: Human tissue collection requires ethical approval and donor consent.
5. Quality Assessment
- Viability Check: Use Trypan blue exclusion assay or flow cytometry with viability dyes.
- Purity Assessment: Confirm by flow cytometry or microscopy.
6. Sterility and Handling
- Work in sterile conditions to avoid contamination.
- Use sterile reagents and disposables.
- Maintain aseptic technique throughout.
7. Downstream Applications
- Cell culture and expansion.
- Genomic, transcriptomic, or proteomic analysis.
- Functional assays, including infection or drug-response studies.