SCH1111 Fundamental Biomedical Techniques: Cell Culture & Stem Cells

Topic = Cell Culture and Stem Cells

Overview of Cell Culture

• Definition of Cell Culture: It is the process by which cells are grown outside of a living organism under strictly controlled conditions.

• Practical Context: In common laboratory practice, the term refers specifically to the culturing of cells derived from animal sources.

• Historical Milestone: The process was first successfully undertaken by Ross Harrison in 1907. He developed a culture to study the development of nerve fibers, using a frog as the source. Harrison documented nerve fibers after $59\,\text{hours}$ in culture.

Essential Cell Culture Equipment

• Cell Culture Hood: A laminar flow cabinet used to maintain an aseptic environment for handling cells.

• Incubator: Typically set to maintain a temperature of $37^\circ\text{C}$ and a concentration of $5\%\,\text{CO}_2$.

• Water Bath: Used for warming media and reagents to appropriate temperatures before use.

• Centrifuge: Used for concentrating cells or removing old media/reagents from cell suspensions.

• Refrigerators and Freezers: Used for storage of media and reagents, typically at $-20^\circ\text{C}$.

• Cell Counters: Tools used to determine cell density and viability, such as an Automated Cell Counter or a Hemacytometer (e.g., Neubauer Improved mechanism with a depth of $0.100\,\text{mm}$ and area of $0.0025\,\text{mm}^2$).

• Inverted Microscope: Specifically designed with the light source above and objectives below to view cells growing on the bottom of a culture vessel.

• Liquid Nitrogen Storage: Used for long-term cryopreservation of cells at temperatures below $-130^\circ\text{C}$. Liquid nitrogen itself is at $-195.79^\circ\text{C}$.

• Autoclave: Required for sterilizing equipment and waste through high-pressure steam.

Applications of Cell Culture

• Basic Cell Biology Models: Used to study cell interactions, effects of disease-causing agents, drug effects, and the processes/triggers of aging and nutrition.

• Toxicity Testing: Evaluating the effects and safety of new drugs on cellular systems.

• Virology: Cultivation of viruses for vaccine production and the study of infectious cycles.

• Cancer Research: Studying how chemicals, viruses, and radiation convert normal cultured cells into cancerous (transformed) cells.

• Genetic Engineering: Large-scale production of commercial proteins and viruses for vaccines (e.g., Polio, Rabies, Chicken Pox, Hepatitis B, and Measles).

• Gene Therapy: Replacing cells containing non-functional genes with cells carrying functional genes.

• Tissue Engineering: Using cell and tissue culture to generate artificial tissues and organs for transplant or study.

Primary Cell Culture

• Definition: Cells taken directly from animal tissue and added to a culture medium.

• Physiological Relevance: Primary cells more closely mimic the physiological state of cells in vivo compared to established cell lines, providing data that is more representative of living systems.

• Finite Life Span: Primary cultures only divide for a limited number of population doublings before reaching senescence.

• Senescence: The state where cells stop dividing but may remain metabolically active.

• Culture Conditions:

  • Temperature: Standardized at $37^\circ\text{C}$.

  • Atmosphere: $5\%\,\text{CO}_2$.

  • Media: Must contain pH buffers, nutrients, and growth factors.

Components of Culture Media

• Bulk Ions: Components including $Na$, $K$, $Ca$, $Mg$, $Cl$, and $P$. Bicarbonate or $\text{CO}_2$ are used as buffers.

• Trace Elements: Essential elements such as Iron ($Fe$), Zinc ($Zn$), and Selenium ($Se$).

• Sugars: Glucose is the most common energy source used.

• Amino Acids: Includes $13$ essential amino acids.

• Vitamins: Necessary cofactors for cellular metabolism.

• Membrane Integrity Agents: Includes Choline and Inositol for cell structure maintenance.

• Antibiotics: Optional agents used to control bacterial and fungal contamination; they are not required for actual cell growth.

• Serum (Fetal Bovine Serum / FBS): A critical additive containing:

  • Growth-promoting activities and peptide hormones/hormone-like growth factors.

  • Protease neutralizers.

  • Buffering agents for toxic nutrients.

  • Substances that influence the interaction between cells and the substrate.

Techniques for Establishing Primary Cultures

• Explant Cultures:

  • Small pieces of tissue are attached to a glass or plastic vessel using plasma clots or fibrinogen.

  • The vessel is immersed in medium.

  • After several days, individual cells migrate from the tissue explant onto the vessel surface to begin dividing. Evidence shows Neural Crest Stem Cells emigrate from hair follicles bulge at $6$, $8$, and $10\,\text{days}$.

• Enzymatic Dissociation:

  • Mechanical disruption: Tissue is broken up using scissors and forceps.

  • Enzymatic treatment: Fragmented tissue is treated with proteolytic enzymes (e.g., Trypsin and Collagenase) to destroy the extracellular matrix and adhesion proteins.

• Cell Separation Methods:

  • Flow Cytometry / FACS (Fluorescence-Activated Cell Sorting): Uses fluorochrome-labeled antibodies and lasers to sort specific cell types based on light deflection and charge.

  • Magnetic Separation (MACS): Uses magnetic beads coated with specific antibodies (e.g., $\alpha7$-integrin or $CD34$ for endothelial cells) to isolate purified cell populations.

Cell Lines and Strains

• Terminology Transition: A primary culture becomes a "cell line" after the first subculture.

• Finite Cell Lines: Possess a limited life span, typically allowing for $2$ to $100$ divisions before senescence.

• Continuous (Immortalized) Cell Lines: Cells that have been transformed under laboratory conditions or derived from tumors. They can proliferate indefinitely if provided with fresh medium and space.

• Cell Strains: A subpopulation of a cell line positively selected (e.g., via cloning) for specific traits. Strains often undergo genetic changes (e.g., transfection) making them more or less tumorigenic than the parent line.

• HeLa Cells Case Study:

  • 1951: Biopsy taken from Henrietta Lacks without her knowledge.

  • 1953: Supplied to Jonas Salk for Polio vaccine trials.

  • 1955: First human cells successfully cloned by Theodore Puck and Philip I. Marcus.

  • 1966: Ethical concerns raised regarding cancer research on unwitting subjects, leading to NIH internal review boards.

  • 1989: Used by German virologists to link HPV to cancer (Nobel Prize discovery).

  • 2013: Genome sequenced without family consent; later policy established for controlled access after meetings with the Lacks family.

Cell Morphology Types

• Lymphoblast-like: Cells do not attach to the surface; they remain in suspension with a spherical shape.

• Epithelial-like: Cells attach to the substrate and appear flattened and polygonal.

• Fibroblast-like: Cells attach to the substrate and appear elongated and bipolar.

Culture Management and Contamination

• Aseptic Technique: Mandatory use of culture hoods to minimize microbial contamination.

• Types of Contamination:

  • Fungi/Bacteria: Evidenced by turbidity, color changes (pH drop), or visible colonies.

  • Mycoplasma: Difficult to detect; requires PCR or enzymatic tests.

  • Cross-contamination: Over $15\text{--}20\%$ of experiments are conducted with misidentified or cross-contaminated cells. Only approximately $50\%$ of researchers verify cell identity regularly.

• Confluency: The percentage of the surface area covered by cells.

  • Optimal Confluency: $70\text{--}80\%$ is ideal for subcultivating (splitting).

  • Too Low: Cells enter a lag phase and fail to proliferate.

  • Too High: Cells undergo contact inhibition or pile up in tumor-like formations.

• Subculturing Reagents:

  • Trypsin: Cleaves peptide bonds (Lysine or Arginine) in fibronectin.

  • EDTA: A chelating agent that binds Calcium ($Ca^{2+}$) ions, which otherwise inhibit trypsin and maintain cell adhesion.

• Passage Number: The count of how many times cells have been removed from a plate and re-plated.

• Hayflick's Phenomenon: The observation that cells divide for a limited number of passages. This number decreases when cells are harvested from older individuals.

Cryopreservation

• Process: Cooling cells to below $-130^\circ\text{C}$.

• Problem: Ice crystals can puncture cell membranes, causing death.

• Cryoprotectant: Dimethyl sulfoxide (DMSO) is used to protect cells by partially solubilizing the membrane (making it flexible) and interrupting the ice lattice.

• Warning: DMSO is toxic; therefore, thawing must be done rapidly to remove cells from the DMSO solution as quickly as possible.

Stem Cell Biology

• Core Properties:

  1. Immature/Unspecialized: They do not perform specific tissue functions.

  2. Self-renewal: The ability to reproduce themselves through division.

  3. Differentiation: The ability to mature into specialized cell types (e.g., liver, skin, nerve cells).

• Potency Levels:

  • Totipotent: Can form all cell types (e.g., zygote or morula).

  • Pluripotent: Can form many cell types, specifically the three germ layers (e.g., Inner Cell Mass of the blastocyst; ESCs).

  • Multipotent: Can develop into a limited range of cell types within a lineage (e.g., adult stem cells like hematopoietic or mesenchymal stem cells).

  • Oligipotent: Progenitor cells capable of forming only a few types.

  • Unipotent: Differentiated cells that can only produce their own type.

• Division Patterns:

  • Symmetric Division: Produces two identical stem cells or two identical progenitor cells.

  • Asymmetry Division: Produces one stem cell (self-renewal) and one specialized/progenitor cell.

Specific Stem Cell Types and Applications

• Embryonic Stem Cells (ESCs): Derived from the blastocyst. Applications include treating Parkinson's, Alzheimer's, heart disease, and Leukemia.

• Adult Stem Cells: Multipotent and tissue-specific. Used in wound healing (e.g., Keratinocyte grafts for third-degree burns).

• Induced Pluripotent Stem Cells (iPSCs):

  • Reprogramming: Differentiated somatic cells (e.g., from a skin biopsy) are genetically reprogrammed using factors like $OCT4$, $SOX2$, $KLF4$, and $CMYC$ to an ESC-like state.

  • Applications: Patient-specific disease modeling, drug screening, and gene targeting to repair mutations prior to transplantation.

• Mesenchymal Stem Cells (MSCs): Found in bone marrow and placenta. They produce fat, muscle, bone, and cartilage. They also exhibit anti-inflammatory and immune-suppressing properties.

  • Differentiation Pathways: Regulated by factors such as $BMP\text{-}2/4/7$, $TGF\text{-}\beta$, $FGF$, Insulin, and $Wnt$ signaling to form adipocytes, osteoblasts, chondrocytes, and myocytes.

Analysis of Cultured Cells

• Cell Morphology: Observed via microscopy.

• Genetic Analysis: Measuring gene expression via RT-PCR (Reverse Transcription Polymerase Chain Reaction) after RNA collection.

• Protein Analysis (Immunocytochemistry):

  • Uses the immune system's specificity to generate antibodies against antigens.

  • Primary antibodies bind the protein of interest; secondary antibodies with fluorescent or enzyme labels allow visualization.

  • Example: Neutral stem cells derived from iPSCs express markers like Nestin and $Sox1$. Differentiated neurons express $\beta II\text{-tubulin}$ (green) and astrocytes express Glial Fibrillary Acidic Protein (red), with nuclei stained by DAPI (blue).