Cell Sourcing and Cell Culture - Lecture 5

3 cell sources:

allo (inter-species), auto (self), xeno (animals)

3 cell types to consider for sourcing:

stem, primary, cell lines

Combinations of choices for cell source consideration:

• Isolate cell & transplant

• Isolate cell, increase # & transplant

• Isolate stem cell, increase #, make it into the cell you want & transplant

Answer “okay”

okay

Name the process by which a simple initial structure becomes complex. (Example: successive differentiation of an unstructured egg)

epigenesis

zygote

single hybrid cell, fertilized ovum

a cluster of dividing cells made by a fertilized egg

blastocyst

new cells formed during early cell division (cleavage) — contained by blastocyst

blastomeres

In embryonic development:

“cell-cell interactions create polarization of the cell and organization — functional distinctions begin to exist between cells (16-32) cells”

What are the 2 distinct cell populations at this time?

trophoblasts and inner cell mass

in embryonic development, a thin layer of cells that helps a developing embryo attach to the wall of the uterus, protects the embryo, and forms a part of the placenta. Also provides nutrients to the embryo.

trophoblasts

in embryonic development, the population of cells that becomes the developing fetus

inner cell mass

in the development of germ layers, the process of establishing the 3 definitive germ cell layers of the embryo

gastrulation

what are the three definitive germ cell layers of the embryo?

ectoderm, mesoderm, endoderm

a germ cell layer of the embryo that becomes the neural crest, neural tubes, lens, cornea, and skin. The outermost layer.

ectoderm

a germ cell layer of the embryo that becomes the connective tissues, muscles, and circulatory system.

mesoderm

a germ cell layer of the embryo that becomes the respiratory system, gut, and endocrine system(?)

endoderm

the process by which stem cells divide to make more stem cells, perpetuating the stem cell pool.

self-renewal

3 stages of stem cell differentiation:

stem cell — progenitor/precursor cell (somatic cell) — specialized/mature cell (terminally differntiated)

Potentiality of stem cells:

totipotent, pluripotent, or multipotent

potentiality classification: able to give rise to all embryonic and adult lineages

totipotent

potentiality classification: able to give rise to all cell types in an adult

pluripotent

potentiality classification: able to give rise to multiple cells within a lineage

multipotent

self-renewal of stem cells:

unlimited

3 reasons we care about stem cells:

1. _____-_______ — unlimited source

2. ________ — pluripotent (or at least multipotent)

3. Many lack ________ _____ (Class 1 and Class 2 MHC) and/or have low expression of co-stimulatory markers — possibly less __________.

(MHC class I proteins are found on the surface of almost of all nucleated cells in the body. MHC class II proteins are primarily expressed on antigen-presenting cells, including dendritic cells, macrophages, and B cells)

self-renewal, potential, immunogenic markers, immunogenic

3 sources of stem cells

(in between: neonatal, fetal, iPSC)

embryonic, adult, mesenchymal

cells that develop into connective tissue, blood vessels, and lymphatic tissue

mesenchymal stem cells

Example: Myocardial Infarction Clinical Trials

Transplantation of cells to fix damaged heart tissue

“While Phase 1 early trials were promising, high heterogeneity of outcomes were seen for Phase 2 trials (or multi-center) (further high heterogeneity of animal studies)”

What are some potential reasons for this variation?

Delivery of cells (IV to direct injections), “matching” of cells, source of cells, age of cells

Example: Myocardial Infarction Clinical Trials:

“The exact mechanisms of cardiac repair by transplanted are merely unknown. Two main hypotheses exist:”

1. Direct cardiomyogenic/vasculogenic ____________

2. Indirect stimulation of the reparative response through ______ _______.

To test, trace implanted cells — no localization of cells (~1-3% remain/graft), whether injected IV, within the coronary artery, or within the heart tissue itself!

Transplanting dead cells produces the same effect as live cells!

differentiation, paracrine effects

________ _______ _______ (_____) cells are generated via genetic reprogramming of adult somatic cells that have limited differentiation potential but, upon reprogramming, express genes that enable them to regain plasticity and give rise to all cell types.

induced pluripotent stem (iPS)

How many different types of mature cells exist in the human body?1

over 200

• Connected 2D sheets of cells

• Cuboidal morphology

• Polar — basal & superior

epithelial cells

• Alone or loosely connected cells

• Bipolar or spindle morphology

• Migrate individually

• Can differentiate into specialized mesenchymal cells (AIC)

Functions of epithelial cells

covering/protection (e.g. skin), absorption (e.g. gut), secretion (e.g. glands)

Structure of epithelial cells

• Single cell layer

  • Simple squamous (flattened)

  • Simple cuboidal (square-shaped)

  • Simple columnar (taller than wide)

• Multiple cell layers

  • Stratified squamous (flattened)

  • Stratified cuboidal (square-shaped)

  • Pseudo stratified columnar (taller than wide)

Answer: “okay”

okay

Name the type of tissue:

Structure:

• Comprised of cells and ECM (amorphous like hyaluronan and fibrous like type I collagen) in different arrangements and compositions)

Function

• Support (e.g. bones)

• Connections (e.g. tendons & ligaments)

• Protection (e.g. fat pads)

• Storage of fluid (e.g. blood, fat)

connective tissue

Name the type of tissue:

Function:

• Specialized proteins, actin and myosin, allow for cellular shortening and contraction of tissue.

muscle tissue

What are the 3 types of muscle tissue?

skeletal, cardiac, and smooth muscle

Name the type of muscle tissue:

• Attached to bones (induces movement)

• Long multinucleated cells or myofibers; appears striated

skeletal muscle

Name the type of muscle tissue:

• heart (propels blood through circulation)

• single nucleated cells, electrically connected, appears striated

cardiac muscle

Name the type of muscle tissue:

• blood vessels, skin, internal organs

• Squeeze substances through organs

• Single nucleated cells, striated

smooth muscle

Which type of muscle is multinucleated?

skeletal

Nervous Tissue

• Neurons

  • Conducting cell; transmits _____ _______

  • Doesn’t divide (aka _________)

  • Dendrites

    • Short and branching processes

    • Increased surface area to receive signals

    • Transmit impulses to neuron ____ _______

  • Axons

    • Only single process

    • Some axons surrounded by ______ (aids in conductance)

    • carry impulses away from cell body

• _________ (second cell type)

  • Nonconductive

  • Support, nourish, and protect neurons

electrical impulses, amitotic, cell body, myelin, neuroglia

Name the type of tissue:

“polarized epithelial cells that produce proteins from other molecules surrounding these cells”

glandular tissue

what are 2 types of glands?

exocrine, endocrine

What type of gland is this?

“secrete products into body cavities or onto body surfaces via ducts”

• Many kinds of duct structures

• Modes of secretion

exocrine glands

what type of gland is this?

“secrete hormones directly into the blood”

endocrine glands

Organs are composed of repeating functional subunits.

Organs in the body are made up of multicellular functional subunits that perform specific organ functions.

Answer: “okay”

okay

culture derived from dispersed cells taken from original tissue, from a primary culture, or from a cell line by enzymatic, mechanical, or chemical disaggregation.

cell culture

Cell culture metholodologies:

1. Cell _______

2. _______ of cells outside the body

3. _______ or expansion in vitro

isolation, maintenance, multiplication

Why would we want to culture cells outside of the body?

1. Understand how cells behave in controlled environment

2. Toxicology (study the effects of drugs on cells)

3. Production of biologics (e.g., monoclonal antibodies, hormones, enzymes, vaccines, etc…)

Answer: “okay”

okay

What is toxicology?

the effect of drugs on cells

what are 3 types of primary culture?

cell culture, primary explants, organ culture

the culture of small pieces of tissue surgically removed from animal tissue or organ. Pieces of tissue are seeded on a substrate to encourage the spreading and outgrowth of the epithelial cells.

primary explants

culture in which the tissue is maintained to preserve its original structural and functional form. The maintenance or growth in vitro of organ primordia or all or part of an organ in a way that may allow differentiation and preservation of the architecture and/or function.

organ culture

Benefits of cell culture:

• Can isolate specific, individual cell types

• Can control _________ conditions

• Free of systemic ________ between animals

• Easy to access the cells

• Usually ______ quantities can be obtained (isolation or expansion)

• Usually (more/less) expensive than animal studies

environmental, variations, large, less

Drawbacks of cell culture

• Environment is not the same as in the animal

• Monolayer is not 3D; no 3D tissue _______

• Complex in vivo signaling environment (present/absent)

architecture, absent

What cell isolation technique is being described?

1. Dissect piece of tissue of interest (biopsy)

2. Cut into small pieces

3. Place pieces on plastic dish & cover with liquid medium

4. Place cells in physiologic environment (37 C, pH 7.4)

5. Allow sufficient time for cells to migrate from tissue

explant technique

Benefits of explant technique for cell isolation:

• easy, (uncomplicated/complicated)

• cells are (harmed/unharmed) by mild process

uncomplicated, unharmed

Drawbacks of explant technique for cell isolation:

• Migration takes a (short/long) time

• Only _______ cells are obtained

• (Low/high) # of cells / mass of biopsy

long, migrating, low

what cell isolation technique is being described?

Method 1

1. Numerous cell adhesions to ECM require calcium — chelate calcium

2. Perfuse with a calcium chelator such as EDTA (EDTA is a chemical that binds certain metal ions, such as calcium, magnesium, lead, and iron)

3. Mechanically disrupt tissue

Method 2

1. Enzymatic dissociation w/ proteases

2. Rinse tissue of blood (can inactivate proteases)

3. Incubate or perfuse w/ enzymes

4. Allow sufficient time for ECM to break down and free resident cells

chemical disassociation

Benefits & Drawbacks of chemical dissociation: enzymatic dissocation

• (fast/slow)

• isolate (few/all) cells

• ______ exposure can damage cells

• _________ inconsistent

fast, all, enzyme, proteases

Cell culture maintenance

Vital environmental conditions:

• ______ pH

• ____ degrees Celsius

• correct ion balance (_______)

• ______ supply

neutral, 37, isotonicity, oxygen

what 2 soluble factors are involved in sustaining cell metabolism?

tissue culture medium, serum

What does tissue culture medium provide?

nutrients; sugars, amino acids, vitamins, minerals

what does serum provide?

assortment of proteins and hormones normally found in blood

How much serum is usually added to a cell culture?

1-20% by volume of media

what can you substitute for serum?

specific growth factors and hormones

Insoluble factors involved in sustaining cell metabolism:

• Insoluble factors = adhesion matrix

• Most cells are ________ dependent

• What cells to attach to (along w/ soluble factors)

  • Controls their function

• Proteins attach to glass/plastic

  • Cells attach to _______

anchorage, proteins

Why are cells usually cultured in incubators with a CO₂ supply of 5-10% with sodium bicarbonate in the medium?

To buffer the media to pH 7.4

An increase in CO₂ in the media leads to a pH (increase/decrease), making it more (basic/acidic).

H₂O + CO₂ —> H₂CO₃ —> H⁺ + HCO₃^-

decrease, acidic

What are 2 ways O₂ delivery is regulated in cell culture?

1. K, ________ of O₂ in media at ____ degrees Celsius

2. ________ of O2 from _____ phase to cell surface

solubility, 37, transport, gas

What drives O₂ diffusion?

(according to Fick’s Law)

concentration gradient

what is the relationship between uptake rate and diffusion rate at steady state conditions?

uptake rate = diffusion rate

How is steady state O₂ tension at cell surface regulated?

1. ______-phase oxygen ________, P_g

2. _______ of the media, d

gas, tension, thickness

increasing the number of cells (also referred to as cell expansion or proliferation)

growing cells

what are the three steps in growing cells?

plating or seeding, proliferation, passaging

Growing cells step 1: Plating or seeding

• Place solution w/ cells, known as a ________, into culture container

• ________ forces cells to make contact w/ container surface where they can ________

suspension, gravity, adhere

growing cells step 2: proliferation

• Cells start at (low/high) density —> as the cells divide, their density per area increases

• Most normal cells (non-cancerous) are _______ inhibited — proliferation stops when area covered

low, contact

growing cells step 3: passaging

• Perform when cells cover almost all dish area

• Use _________/_________ to release cells

• Plate the cells again at a (lower/higher) density (dilution)

• Cells can then proliferate again

• P0 = primary isolation; P1 - 1st pass; P2 = 2nd pass

• Some cells (e.g., neural) do not grow in culture

proteases/enzymes, lower

What is described below?

“the majority of cells can only divide a specific number of times and then they die. This limits how much one can amplify cell number.”

Hayflick limit

What are 3 causes that can transform or immortalize cells?

These cells are unlimited in their growth potential.

They can be obtained from ______ tissue and are referred to as ______ _______.

Since they do not exhibit normal growth behavior, they can be dangerous to employ in tissue engineering.

radiation, chemicals, spontaneous changes, cancer, cell lines

what are 4 parameters involved in tailoring tissue culture environment to mimic in vivo state?

soluble factors, matrix interactions, cell-cell interactions, physical factors

What does a characteristic cell growth curve look like? (no passing)

Answer: “okay”

okay

systemic factors present in the circulation in vivo

soluble factors

What is a strategy to replace soluble factors in a tissue culture environment?

add appropriate _________/______ ________ or ______ to culture ______

hormones/growth factors, serum, medium

what parameter is related to the following problem?

extracellular matrix composition from native tissue required for cell growth/function

matrix interactions

What is a strategy to replace matrix interactions in a tissue culture environment?

use complex ____ materials

ECM

what parameter corresponds to the following problem?

homotypic and heterotypic cell-cell interactions present in the native tissue are lacking.

cell-cell interactions

What is a strategy to replace cell-cell interactions in a tissue culture environment?

use appropriate _____ _______, _______ with other cells

seeding density, coculture

what parameter corresponds to the following problem?

ECM geometry from native tissue differs from that in vitro, cells in vivo are subjected to physical forces

physical factors

What is a strategy to replace physical factors interactions in a tissue culture environment?

seed cells in ____ structures, subject cultured cells to ______ ______ (e.g. fluid shear stress and mechanical stretch)

3D, physical forces

what are the 3 stages of growth for primary culture/cell line (non-transformed)?

1. Slow growth for primary culture

2. _______ growth stage (linear on log scale)

3. Growth stops (_______ limit) —> senescence & cell death

logarithmic, Hayflick

the process by which a cell ages and permanently stops dividing but does not die

senescence

Autologous Cell Source:

• From _______

• Immune Response: _____/_______

• Supply: varies from patient to patient and cell type

• Time until available for use: ____-_______

• Cost: (low/moderate/high)

• Potential function: match

patient, low/none, days-weeks, high

Allogeneic Cell Source:

• From ____ _______

• Immune Response: _____ (need __________)

• Supply: varies from patient to patient and cell type

• Time until available for use: _______ available

• Cost: (low/moderate/high)

• Potential function: match

same species, some, immunosuppression, immediately, moderate

Xenogeneic Cell Source:

• From ______ _______

• Immune Response: ______ (need _______)

• Supply: good

• Time until available for use: _________ available

• Cost: (low/moderate/high)

• Potential function: ?

different species, strong, immunosuppression, immediately,moderate

3 paths of basic cell decision tree:

grow, chill/quiescent, die

What phase of the cell cycle is this?

gap between mitosis (M phase) and DNA synthesis (S phase)

G1

What phase of the cell cycle is this?

DNA replication

S phase

What phase of the cell cycle is this?

cell volume increase prepping for division

G2 Phase