Tissue Engineering Quiz 3

Cell culture

process by which cells are grown under controlled conditions, generally outside of their natural environment

organoid culture

a tissue culture method to grow functional 3D organoids from a group of cells and combination of various biochemical factors

organ culture

a development from tissue culture methods of research, the organ culture can accurately model functions of an organ in various states and conditions using the actual in vitro organ itself

Why is so much research carried out on cultured cells?

can be obtained in large quantities, typically consist of a single cell type, allow for the study of various cellular activities, can differentiate, and respond to treatments with drugs, hormones, and growth factors

Goal of cell culture

maintain & expand population of cells; focus on cell viability

What is cell theory?

organisms made of 1+ cells, cell is structural unit of life, cells arise from division of other cell, DNA is passed from cell to cell

primary culture

started from cells, tissues or organs taken directly from ananimal

explant

an excised fragment of an organ which usually retains some degree of tissue architecture

tissue culture

the maintenance of a tissue or fragment thereof in a way that may allow differentiation and preservation of the architecture and/or function

organoid

an artificially grown mass of cells or tissue that resembles an organ

monolayer

single layer of cells growing on a surface

subculture

the transplantation of cells from one culture to another (passaging)

cell line

arises from the primary culture at the time of the first subculture ‐ "finite" life span

Primary Cell Lines

isolated directly from animal or plant tissue

Continuous Cell Lines

a finite cell line undergoes transformation and acquires the ability to divide indefinitely

Transformed Cell Lines

from tumors or are transformed either spontaneously or deliberately

What are suspensions cells?

able to survive and proliferate without attachment to the culture vessel and are typically derived from sources like blood, spleen, and bone marrow

What are the advantages of suspension cells?

large numbers and ease of harvesting

What are adherent cells?

grow in a monolayer and are attached to the surfaces of culture vessels, typically derived from ectodermal or endodermal embryonic cells

What are the advantages of adherent cells?

ability to spread on surfaces, making them easy for microscopy or other functional assays

Anchorage‐dependent cells

require attachment to the surface for cellproliferation

Anchorage‐independent

property of transformed cells

Maintenance

examine daily cultures, observe the morphology, the color of the medium and the density of the cells

Feeding in suspension cultures

dilution into fresh medium

Feeding in adherent cultures

replace old with fresh medium

Splitting

Done at 75 ‐100% cell confluence, prevents the increase in cell death and prevents the decrease in mitotic index

What can happen to cells during storage?

can be damaged by ice crystals, electrolyte changes, dehydration, and pH shifts

What can be added to prevent damage to cells in storage?

cryoprotective agent can be added to lower the freezing point

Requirements for cell culture

cells, sterile env, controlled growth env, space for growth, food

What is the most commonly used mammalian hosts for industrial production of recombinant protein therapeutics?

CHO (Chinese Hamster Ovary Cells)

Solid substrates

support cell growth in monolayers for expansion

Semisolid substrates

support 3D structures and tube formation

Liquid substrates

used for growing cells in suspension

Feeder layers

provide a base for culturing other cells

After the first subculture, the primary culture becomes...

cell line

Cell Strain

subpopulation of a cell line selected from the culture through cloning or other methods, often acquiring additional genetic changes

Senescence

normal cells lose the ability to divide after a limited number of times

Substrate must provide...

Growth factors, Hormones, Gases (O2, CO2), A regulated physico-chemical environment

Why is cryopreservation used?

to store surplus cells long-term by treating them with a protective agent and keeping them cool until needed.

morphology

dividing cells in culture based on shape/appearance

Fibroblastic cells

grow attached to a substrate; bipolar/multipolar and elongated

Epithelial-like cells

grow attached to a substrate in discrete patches; polygonal

Lymphoblast-like cells

grown in suspension without attaching to a surface; spherical

What gases are added into the culture environment?

O2 and CO2

Why is carbon dioxide (CO₂) used in cell cultures?

helps regulate pH in bicarbonate-buffered media; can be incorporated into ribose for research purposes

What does culture media provide for cell growth?

nutrients, buffering, amino acids, salts, glucose, serum and isotonic conditions tailored to each cell type

Initial cell culture attempts used "natural media" based on...

tissue extracts and bodily fluids

What is serum?

clear liquid from blood that supplies nutrients, growth factors, hormones, and attachment factors for cell culture

What are the disadvantages of using serum in cell culture?

undefined composition, high batch variability, potential contamination, and complicates protein purification

Disadvantages Of Serum‐free

highly selective, different media for different cells, slower proliferation, lack of reagent purity

What factors influence the optimal temperature for cell culture?

animal's body temperature, regional variations, and the need for consistency

What types of contamination can affect cell cultures?

bacteria, yeasts, fungi, molds, and mycoplasma

Sources of contamination

lack of sterile technique, cells, air supply

Signs of contamination

pH changes, cloudy medium, culture appearance change, etc

How to prevent contamination

sterile techniques, antibiotics, fungizides

What are tissue culture flasks?

sterile containers made of polystyrene, treated to help cells attach and can be coated with substances to promote growth

What is a biological safety cabinet?

protects users from harmful biological agents while working with materials

What is a horizontal laminar-flow clean cabinet?

not useful for hazardous organisms but best protection for cultures; blows filtered air across the workspace from the back to the front to keep it sterile

What is a vertical laminar-flow clean cabinet?

best for working with hazardous organisms; blows filtered air down onto the workspace to maintain a sterile environment

What is an incubator?

controlled environment used to grow and maintain biological cultures by providing optimal temperature, humidity, and gas levels

What are indicators in culture media?

show pH changes without affecting most biological functions, but may interfere with some bioassays (ex: phenol red)

What are balanced salt solutions?

made of inorganic salts, sodium bicarbonate, and sometimes glucose, foundation for many media and contributing to osmolality

What is antibiotic resistance?

bacteria survive antibiotics, making it hard to control contamination in cell cultures

What is passaging of cells?

splitting adherent cells in culture vessels, often needed every few days to keep them healthy

What is subculture in cell culture?

process of detaching proliferating adherent cells from a dish, diluting them, and reseeding them into fresh vessels, turning them into a cell line

What does forming a cell line from primary culture imply?

increased cell numbers, dominance of fast-growing cells, and uniformity in the cell population

What is aseptic technique?

practices to prevent contamination from pathogens and minimize infection risk

Why is 70% alcohol used for disinfection?

slows protein coagulation, allows better penetration into cells, and includes water, which helps denature proteins and enhances its antimicrobial effects

What are selective inhibitors used for in cell culture?

help isolate specific cell types by allowing only certain cells to grow

What role does D-valine play in purifying cell cultures?

replaces L-valine in the medium, allowing cells with D-amino acid oxidase to grow preferentially, aiding in cell isolation

How can cell separation be achieved in cultures?

centrifugation to increase cell density, or using antibody-based techniques to target specific cell types

What is the purpose of transfection in cell culture?

introduces drug resistance to a subpopulation of cells, allowing researchers to isolate these cells by adding the drug to the culture medium

How does centrifugation aid in cell separation?

increases cell density by sedimentation, allowing for the separation of different cell types based on their density

What are antibody-based techniques used for in cell culture?

used to isolate specific cell types by targeting unique antigens on their surface

Why is primary culture often yields...

heterogeneous population of cells

How can cells lose their functionality in vitro?

de-differentiation

Explain differentiation

process leasing to expression of phenotypic properties characteristic of the functionally mature cell in vivo

two-dimensional culture system

cells are grown on the flat surface of a dish

three-dimensional culture system

cells are grown in a 3D matrix consisting of extracellular materials; better suited to study cell-cell interactions

How To Characterize a cell line?

using antibodies for specific antigens, enzymes, and RNA/DNA fingerprinting

How are antibodies used to identify specific cell types?

target unique antigens allowing identification based on cell-specific surface, cytoplasmic, or nuclear markers

What is an antibody?

immunoglobulin that specifically binds to the antigen that triggered its production

What are antibodies made of?

glycoproteins made of two heavy and two light chains, which together form the antigen-binding site

What is the fragment antigen binding (FAB) of an antibody?

binds to an antigen and includes one constant and one variable domain from each heavy and light chain

What makes each antibody unique?

variable regions of their light and heavy chains differ based on DNA arrangement

What is somatic recombination?

certain DNA is removed from the antibody coding region, creating antibody diversity

What is an antigen?

substance that triggers an immune response and binds to the antibodies produced against it, usually being a protein, polysaccharide, or sometimes a nucleic acid

What is an epitope?

specific site on an antigen recognized by an antibody, typically made up of a few sugars or amino acids

Where are antibodies produced?

vertebrate cells

How are antibodies produced?

macrophages engulf foreign molecules, displaying their parts on surface --> activates B-cells to produce antibodies

Why are antibodies produced?

bind to and mark foreign substances for destruction, helping the immune system eliminate them

How are monoclonal antibodies produced?

hybridoma, which is made by fusing an antibody-producing cell with a myeloma cell, allowing large-scale antibody production

What is unique about monoclonal antibodies' recognition?

recognize only a single specific epitope on an antigen

How precise are monoclonal antibodies in distinguishing proteins?

can tell the difference between two polypeptides that differ by just one amino acid

How are polyclonal antibodies produced?

immunize animal with antigen -> collect its serum -> purify the antibodies from it

Why are polyclonal antibodies considered a mixture?

one antigen stimulates the production of multiple antibodies in response

What's more expensive: polyclonal or monoclonal antibodies?

monoclonal

Monoclonal traits compared to polyclonal

Mouse or rabbit hybridoma, cleaner, consistent, more likely to get false negative results

Polyclonal traits compared to monoclonal

Many different species, non-specific reactivity, varied avidity/affinity, more likely to have success in an unknown application

How do secondary antibodies work?

attach to the heavy chain of primary antibodies and contain detection markers