Cell Culture Basics
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Questions and Answers

What is the primary difference between primary cells and immortalized cells?

  • Immortalized cells are taken directly from living tissue.
  • Primary cells are harder to culture than immortalized cells. (correct)
  • Primary cells can divide indefinitely.
  • Immortalized cells are derived from living tissue.

    • Cell lines derived from primary cultures have an indefinite life span.

    False

    What term is used to describe the process of transferring cells to a new vessel for continued growth?

    subcultured

    Primary cells are obtained by dissociating tissue fragments _____ to single cells.

    <p>enzymatically</p> Signup and view all the answers

    Match the cell lines to their common uses:

    <p>HEK = Human embryonic kidney cells A549 = Human lung carcinoma cells Jurkat = Human T lymphocyte cells Vero = African green monkey kidney cells</p> Signup and view all the answers

    Which of the following cell lines is commonly used and originates from cancerous tissue?

    <p>A549</p> Signup and view all the answers

    Immortalized cells can be derived from normal tissues.

    <p>False</p> Signup and view all the answers

    What is the term for the growth of cells in a controlled environment outside of their natural conditions?

    <p>cell culture</p> Signup and view all the answers

    What is the purpose of using media without phenol red in assays?

    <p>To prevent interference in fluorescence experiments</p> Signup and view all the answers

    Fetal bovine serum (FBS) is a defined mixture of nutrients and growth factors.

    <p>False</p> Signup and view all the answers

    What temperature and duration are required to heat inactivate serum for cell culture?

    <p>56°C for 30 minutes</p> Signup and view all the answers

    Serum provides various growth factors, including __________ and __________.

    <p>fibroblast growth factor (FGF), epidermal growth factor (EGF)</p> Signup and view all the answers

    Match the following components with their roles in cell culture:

    <p>Albumin = Carries vitamins and hormones Transferrin = Carries iron Fibronection = Promotes cell adherence Antibiotics = Prevents bacterial growth</p> Signup and view all the answers

    Which component in serum helps to neutralize proteases released by certain cells?

    <p>Anti-protease ingredients</p> Signup and view all the answers

    Including antibiotics in cell culture is unnecessary when aseptic techniques are applied.

    <p>False</p> Signup and view all the answers

    Name one vitamin that acts as a coenzyme in cell metabolism processes.

    <p>Any vitamin (e.g., vitamin B complex)</p> Signup and view all the answers

    Which condition can be treated with stem cell transplants from cord blood?

    <p>Leukemia</p> Signup and view all the answers

    Cord blood stem cells are only used for research purposes.

    <p>False</p> Signup and view all the answers

    What is cryopreservation?

    <p>A method of freezing and storing cells at very low temperatures.</p> Signup and view all the answers

    Which medium is suitable for fast-growing tumor cells and contains iron and phenol red?

    <p>DMEM</p> Signup and view all the answers

    The cryoprotectant used in cryopreservation helps prevent the formation of __________.

    <p>ice crystals</p> Signup and view all the answers

    RPMI-1640 is primarily used for mouse kidney cell cultures.

    <p>False</p> Signup and view all the answers

    Match the following conditions with their corresponding treatments using cord blood:

    <p>Leukemia = Stem cell transplant from cord blood Anemia = Stem cell transplant from bone marrow Sickle cell disease = Stem cell transplant from cord blood Parkinson's disease = Research for treatment using cord blood</p> Signup and view all the answers

    What is primarily used as a cryoprotectant in the freezing medium?

    <p>DMSO</p> Signup and view all the answers

    What are the two main categories of cell culture contaminants?

    <p>Chemical contaminants and biological contaminants.</p> Signup and view all the answers

    Cryopreservation can minimize genetic change in stem cells.

    <p>True</p> Signup and view all the answers

    DMEM is modified from __________.

    <p>Eagle's Medium</p> Signup and view all the answers

    What is the minimum cell viability required for successful cryopreservation?

    <p>75%</p> Signup and view all the answers

    IMDM is particularly well-suited for which type of cell culture?

    <p>Hybrid cell selection after cell fusion</p> Signup and view all the answers

    Match the following cell culture media with their characteristics:

    <p>DMEM = Suitable for tumor cells with fast growth RPMI-1640 = Used for human lymphocyte cultures IMDM = Used for hybrid cell selection Ham's F-12 = Supports serum-free growth of mammalian cells</p> Signup and view all the answers

    It is impossible to reduce the frequency of cell culture contaminants.

    <p>False</p> Signup and view all the answers

    Name one type of chemical contaminant commonly found in cell culture.

    <p>Endotoxins</p> Signup and view all the answers

    Which cell types can mesenchymal stem cells (MSCs) differentiate into?

    <p>Osteoblasts, Chondrocytes, Adipocytes</p> Signup and view all the answers

    Mesenchymal stem cells (MSCs) are primarily derived from peripheral blood.

    <p>False</p> Signup and view all the answers

    Name one therapeutic application of mesenchymal stem cells (MSCs).

    <p>Tissue repair</p> Signup and view all the answers

    The percentage of cell viability is calculated after counting viable and dead cells in the _____ large quadrants.

    <p>4</p> Signup and view all the answers

    Match the following sources of MSCs with their respective types:

    <p>Bone marrow = Primary source of MSCs Umbilical cord = Source of MSCs Adipose tissue = Source of MSCs Dental pulp = Source of MSCs</p> Signup and view all the answers

    Which of the following is NOT a source of mesenchymal stem cells (MSCs)?

    <p>Spleen</p> Signup and view all the answers

    Hematopoietic stem cells (HSCs) are capable of differentiating into a variety of cell types, including fat cells.

    <p>False</p> Signup and view all the answers

    What is the primary function of hematopoietic stem cells (HSCs)?

    <p>To differentiate into blood cells and immune cells</p> Signup and view all the answers

    What does a flow cytometer primarily measure?

    <p>Fluorescence intensity from labeled antibodies</p> Signup and view all the answers

    Flow cytometry involves passing cells in single file in front of a laser for analysis.

    <p>True</p> Signup and view all the answers

    What is the role of phosphatidylserine (PS) in early apoptosis?

    <p>PS moves to the extracellular membrane.</p> Signup and view all the answers

    During ________, cells exhibit nuclear condensation and DNA fragmentation.

    <p>apoptosis</p> Signup and view all the answers

    Match the following terms to their descriptions:

    <p>Necrosis = Loss of membrane integrity and cell swelling Apoptosis = Cell death process involving nuclear condensation Annexin V = Binds to externalized phosphatidylserine Propidium iodide = Viability dye that penetrates damaged cells</p> Signup and view all the answers

    Which of the following is a characteristic feature of necrosis?

    <p>Loss of membrane integrity and cell lysis</p> Signup and view all the answers

    Late-stage apoptosis is marked by the loss of membrane integrity and the ability of PI to penetrate the cell.

    <p>True</p> Signup and view all the answers

    What is the function of sheath fluid in flow cytometry?

    <p>To focus the cell suspension.</p> Signup and view all the answers

    Study Notes

    Stem Cell Biology (BIO414) Lab Information

    • Course Coordinator: Dr. Shaza Ahmed
    • Course Assistant: Aya El-Sharkawy
    • Fall 2024

    Lab 1: Introduction to Cell Culture

    • Cell culture involves removing cells from animals or plants & growing them in artificial environments.
    • Cells can be taken directly from tissue or derived from established cell lines.
    • Primary cultures are the initial stage where cells from tissue are isolated and proliferated until they fill the available space.
    • Subculturing (or passaging) is necessary at confluence to provide more room for continued growth.
    • Primary cultures have a limited lifespan.

    Primary vs Immortalized Cells

    • Primary cells are taken directly from living tissue.
    • Immortalized cells are derived from cancerous tissues or cells transfected with oncogenes.
    • Primary cells have a low survival rate and only a few divide.

    Primary cell culture: By cell differentiation

    • Differentiated cell culture; differentiated cells have lost the capacity to differentiate further.
    • Stem cell culture; undifferentiated cells are able to differentiate into other cell types.
    • Monolayer cultures; anchorage-dependent cells grow as a continuous layer on the culture vessel.
    • Suspension cultures; cells grow freely in the medium.

    STEM CELLS CLASSIFICATION

    • Totipotent: Differentiate into any cell type (e.g., zygotes)
    • Pluripotent: Differentiate into cells from any of the three germ layers (e.g., embryonic stem cells, iPSCs)
    • Multipotent: Differentiate into a limited range of cell types (e.g., adult stem cells - mesenchymal or hematopoietic)
    • Oligopotent: Differentiate into a limited number of cell types (e.g., adult stem cells - lymphoid, myeloid)
    • Unipotent: Differentiate into single cell type (e.g., adult stem cells - satellite, epidermal)

    Cell Culture Laboratory Equipment

    • Biosafety Level 2 (BSL-2) lab is required
    • Class II biosafety cabinet (laminar flow hood)
    • CO2 incubator
    • Water bath
    • Centrifuge
    • Refrigerator and Freezer (-4°C, -20°C, and -80°C)
    • Inverted microscope
    • Autoclave
    • Liquid nitrogen or cryostorage container

    Cell Culture Supplies

    • Cell culture vessels (flasks and plates)
    • Assorted sterile pipettes and pipetting devices
    • Cryovials
    • Falcons and Eppendorfs
    • Controlled-rate freezing container (canister)

    Following Aseptic Technique During Cell Culture

    • Sterile work area; cell culture hood, restricted area, free from traffic, UV light sterilization, disinfected with 70% alcohol
    • Good personal hygiene; wash hands before and after handling cultures, wear PPE (gloves, safety glasses, closed-toe shoes, protective clothing)
    • Sterile reagents and media; reagents, media, or solutions must be sterilized with appropriate procedure before use.

    Cell Culture Media Ingredients

    • Carbon source (e.g., glucose)
    • Buffering system (e.g., HEPES)
    • pH indicator (e.g., phenol red)
    • Serum (fetal bovine serum)
    • Metabolites, vitamins, and minerals
    • Antibiotics (penicillin/streptomycin)

    Optimum Culture Conditions

    • Incubated in a sterile incubator
    • Tightly regulated temperature (e.g., water-jacketed incubator) and CO2 concentration
    • Ideal growth conditions are: 37°C, 5% CO2 with saturating humidity

    Basal vs Complete Media

    • Basal media; minimal nutrients, including carbon source, amino acids, and salts, allowing addition of specific nutrients .
    • Complete media; wider range of essential nutrients, including vitamins and amino acids, often used when a large quantity of cells is needed.

    Cell Viability

    • Cells can be counted using a hemocytometer (to determine the cell count and viability).
    • Trypan blue is added to distinguish between live and dead cells.

    Lab 3: Culture of Stem Cells

    • Methods for Obtaining Stem Cells
      • From cell bank or isolating cells from donor tissue
    • Methods to isolate cells from tissue
      • Enzymatic method
      • Explant method
    • Enzymatic Method: Using proteolytic enzymes to detach cells from tissue (dilute or inhibit enzyme reaction).
    • Explant Method: No enzyme required, the tissue is excised and placed in the media to allow cells to migrate.

    Adherent vs. Suspended Cells

    • Adherent cells; grow attached to a solid or semi-solid surface (e.g., tissue culture flasks, plates).
    • Suspended cells; grow freely in a liquid medium (e.g., suspension culture).

    Manipulation of Stem Cells

    • Cell Passaging/Splitting; dividing a confluent culture to provide room.
    • Media Change (Feeding); removing and replacing the culture media.
    • Differentiation; a less specialized cell developing into a more specialized cell type.

    Lab 4: Cord Blood Banking & Cryopreservation

    • Cord blood banking; collecting and storing cord blood after birth.
    • Cord blood is rich in stem cells, which can treat many diseases
    • Stem cells is used for transplants in sick people or medical research.

    Cryopreservation

    • Process of freezing cells at very low temperatures (-200°C) , maintaining their viability .
    • Special freezing medium is used; normal serum-supplemented medium plus cryoprotectant (such as DMSO or glycerol) to prevent ice crystal formation.

    Lab 5: Extraction of Mesenchymal Stem Cells

    • Mesenchymal Stem Cells (MSCs): Fibroblastoid multipotent adult stem cells with self-renewal and differentiation.
    • Sources of MSCs: bone marrow, adipose tissue, umbilical cord matrix.
    • Location of MSCs: perivascular niche.

    Lab 6: Flow Cytometry for Stem Cell Markers

    • Flow Cytometry: A technique to analyze cell populations, properties, and viability.
    • Flow cytometer measures various aspects and components of cells.

    Immunophenotyping

    • Immunophenotyping; labeling cells with fluorescently tagged antibodies; gaining insight into immune response composition

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    Stem Cell Biology (BIO414) Lab 1 - Introduction to Cell Culture PDF

    Description

    Test your knowledge on the fundamentals of cell culture, including the differences between primary and immortalized cells. This quiz covers essential concepts such as cell line usage, growth factors, and environmental conditions for cell growth.

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