Describe the methods to establish primary cell

culture

Discuss the applications of primary cell culture

in biomedical sciences.

Learning outcomes

Cultures established from a specific

organ site with specific processing

Sources Adult- human biopsy

materials, animal tumour

Embryo-mouse, chicks,

organ rudiments

Egg-embryonated

Primary cell cultures typically will have a finite life

span in culture (maximum passage 20-40X)

Continuous cell lines are, by definition, abnormal and

are often transformed cell lines (can be passaged

indefinitely).

"mimic" in vivo conditions more closely than cell lines.

able to accurately model functions of an organ in various states and conditions by the use of the actual in vitro organ itself.

exclude the influence of other organs and of the circulatory and immune system, thus providing the possibility to study direct effects on a cell population.

Advantages

Cell culture- from tissue that is

disaggregated by enzymatic,

chemical, or mechanical methods

Explant culture- from the outgrowth

of migrating cells from a piece of

tissue or from

Organ culture- whole organ or part of

the organ is maintained in culture

Types

Cell culture

Explant culture.

Organ culture

Fat and necrotic tissues are best removed during

dissection. The tissue should be chopped finely with sharp

instruments to cause minimum damage.

Enzymes used for disaggregation should be removed subsequently by gently centrifugation.

The concentration of cells in the primary culture should be much higher than that normally used for subculture, since the proportion of cells from the tissue that survives in primary culture may be quite low.

A rich medium is preferable to a simple medium Embryonic tissue is preferable as it disaggregates

more readily, yields more viable cells and proliferates more rapidly in primary culture than does adult tissues.

Requirements

Methods

Primary

explant/outgrowth Enzymatic

disaggregation

Mechanical

disaggregation

Tissue is finely dissected and

placed into culture as "primary

explants Cells migrate from tissue onto

culture substrate /vessel

Cell will begin to divide and grow

Cells can be harvested

Explant/

Outgrowth

culture

Harvest the cells

migrating out from the

explant

Explant/

Outgrowth

culture

Tissue is finely dissected

Need to disrupt cell-cell adhesion

proteins

Enzymes such as collagenase or

trypsin are used to digest tissue

Cells can be damaged to point of

lysis

Centrifuged to harvest and wash

cells from enzymes

Enzymatic

disaggregation

Types of cells

-Fibroblasts

-Epithelial

-Muscle

-Bone

-Nerve

Enzymatic

disaggregation

Types of enzymes

Trypsin and pronase give the most complete disaggregation but may damage the cells.

Collagenase and dispase give incomplete disaggregation but are less harmful.

Hyaluronidase + collagenase digest the intracellular matrix.

DNase is employed to digest DNA released from lysed cells.

Tissue

Disaggregation

by Collagenase

Enzymatic

disaggregation

Types of Collagenases

Tissue is finely dissected Cells separated using syringing and

vigorous pipetting Pressing tissue into a mesh

Wash cells through sieve Faster than enzymes but less yield

May cause mechanical damage to cells

Mechanical

disaggregation

Sieving Syringing Vigorous pipetting

Mechanical

disaggregation

Types of cells

-Only soft tissues: spleen, thymus, embryonic liver,

embryonic and adult brain, and some human and animal

soft tumors, respond well to this technique.

- Brain - complete disaggregation can be obtained easily,

the viability of the resulting suspension is lower than that

achieved with enzymatic digestion.

Mechanical

disaggregation

Forcing tissue

through sieve with

syringe piston

Drawing tissue into

syringe

Pipetting tissue tissue

fragments up and

down

Isolation of organ

Cold trypsin

Dissection

Enzymatic disaggregation

Mechanical

disaggregation

Fine

dissection/

explant

Collagenase Warm trypsin

Overnight storage, long

incubation

Long

incubation,

complete

medium

Short incubation,

repeated sampling

Dispersed

primary

culture

CELL LINE

Explant Outgrowth

Secondary

explant Culture

Transfer

Subculture

Centrifuge

Resuspend

and seed

Primary Explant

Media- A rich medium, such as Hams F12, DMEM are preferable to a simple medium, such as Eagles MEM,

Serum - fetal bovine (FBS) often gives better

survival than does calf or horse.

Viability of cells- an inverted phase contrast

microscope.

Live cells - phase bright; suspension cells are typically rounded and somewhat symmetrical; adherent cells will form projections when they attach to the growth surface.

Assessed using the vital dye, trypan blue, which is excluded by live cells but accumulates in dead cells.

Cell numbers are determined using a hemocytometer.

Adherent primary culture - nonviable cells are removed at the first change of medium (floating cells).

Cultures maintained in suspension- nonviable cells are gradually diluted out when cell proliferation starts.

Nonviable cells may be removed from the primary disaggregate by centrifuging the cells on a mixture of Ficoll and sodium metrizoate

(e.g., Hypaque or Triosil) [Vries et al., 1973].

The viable cells collect at the interface between the medium and the Ficoll/metrizoate, and the dead cells form a pellet at the bottom of the

tube.

.

Cell Type Tissue Source Reference

Fibroblast Skin, embryonic Han et al., 1993; Lam et al., 1988.

Brain Cells Brain, embryonic

Brain, post hatch

Nicholas et al., 1986; Shafren and Tannock,

1991. Adams, 1965.

Pituitary cell Pituitary gland, post hatch Preze et al., 1987.

Rigmented retinal cell Retina, post hatch Rosenberg et al., 1989.

Liver cell Liver, post hatch Schultz and Mistry, 1981 (surgical);

Legrand and Lemarchal, 1992 (non-

surgical).

Intestinal epithelial cell Intestine Ali and Rernolds, 1996.

Pancreatic cells Pancreas, post hatch Kodoma et al., 1995.

Kidney cell Kidney, embryonic Chomiak et al., 1960; El Zein et al.m 1971.

Thymic cell Thymus, post hatch Lam et al., 1988;

Bursal cell Bursa, post hatch Lam et al., 1988; Nunoya et al. 1991.

Spleen cell Spleen, post hatch Hurk, 1990;

Leukocyte Peripheral blood, post hatch

Bone marrow, post hatch

Hurk, 1990;

Hurk, 1990;

Macrophage Peritoneal exudate Qureshi and Hagler, 1992.

Adipose cell Abdominal fat, post hatch Cryer et al., 1987.

Cartilage cell Sternal cartilage, embryonic

Growth plates, post hatch

Coon and Cahn, 1966.

Rosselot, et al., 1992.

Tendon cell Toes, embryonic Riederer-Henderson et al., 1983.

Bone Cells

_Osteocyte

_Osteoblast

_Osteoclast

Tibiotarsi, embryonic

Calvaria, post hatch

Medulary bone, past hatch

van der Plas and Nijweid, 1992.

Teti et al., 1991a.

Teti et al., 1991b.

Muscle cell Pectoralis major, post hatch

Breast muscle, embryonic

Thigh, embryonic

McFarland et al., 1988.

Armstrong et al., 1990.

Paterson and Strohman, 1972.

Adrenocartical cell Adrenogland, past hatch Rosenberg et al., 1989.

Endothelial cell Fat pads Twal and Leach, 1996.

Testicular cell Testis, embryonic Rombauts et al., 1995.

Granulosa Follicles, laying hen Yoshimura and Tamura, 1988.

Oviduct cell Oviduct, laying quail Kato et al., 1975.

Fundamental

Bioreactor-production of

biological reagents

(monoclonal antibodies)

Diagnostics-isolation

and identification of

agents, diseases,

mechanism

Stem cell research-embryonic

and adult (somatic) stem

cells, reproductive

biotechnology, gene therapy

Applied Research

Tissue engineering

(regenerative

medicine)

Drug discovery- screen for

effects of compounds like

hormones and drugs

Diagnostics

Clinical detection and

isolation of viruses, how

they grow and infect

organisms

Cell-Based

Manufacturing

Large scale

production of

viruses for use

in vaccine

production-

polio, rabies,

chicken pox,

hepatitis B and

measles.

Cells (genetically

enginereed) to

produce proteins

(monoclonal

antibodies,

insulin,

hormones)

Use of Cells as

replacement tissues

and organs

Artificial skins-

treating burns

and ulcers.

Research-

artificial organs

such as

pancreas,

kidney and liver

Embryonic and adult

stem cells

Supply of

replacement

cells and tissues

Gene therapy

Genetically engineer

cells-cells removed

from patient lacking

functional gene-.

Cells grown and

missing or damaged

gene replaced

Tissue

Engineering

Primary rodent

kidney cell

culture

-Propagation of many types

of viruses i.e. adenoviruses

and rabies.

-Studies related to kidney

and diabetic diseases.

-Study of the toxicity of

drugs

Effects of PSC833 on cerebral

glucose metabolism in rat

primary cultures of cortical

neurons and

Astrocytes (Cruz and Wolf,

2001)

(Biochemical Pharmacology 62

(2001) 129139)

2.3. Primary cultures of cortical neurons Neuronal cultures were prepared from the cerebral cortex

of 17-day-old rat embryos as described by Gomeza et al.

[23]. Tissue was cleaned of meninges and dissociated

with papain (7 min, 37, 0.4 mg/mL in a 10-mM

phosphate buffer solution, pH 7.5, with 6 mM glucose)

and subsequently disaggregated by passing through

a Pasteur pipette. After centrifugation (800 g, 5 min),

pelleted cells were resuspended in DMEM containing 10%

FBS, penicillin (50 IU/mL), and streptomycin (0.5 mg/mL)

and plated on poly- L-lysine (10 mg/mL)-coated plates at

a density of 106 cells/ mL. Medium was replaced after 3 hr

by a serum-free medium prepared with DMEM

supplemented as described by Brewer et al. [24]. This

medium was changed every two days up to the sixth day

after seeding, when the experiment was performed. Cells

were maintained at 37, 95% air and 5% CO2. Neuronal

cultures were characterized immunohistochemically using

a monoclonal antibody antineurofilament 160 kD (anti-

NF160) [25]. The monoclonal antibodies antiglial fibrillary

acidic protein (specific for astrocytes) [26], antivimentin

(specific for astrocytes and fibroblast) [27], and

antifibronectin (specific for fibroblast) [28] were used to

verify the purity of the cultures. Neuron enrichment was

higher than 90%. Experiments were performed after cells

were maintained for 6 days in culture.

Susceptibility of primary cultures of proximal tubular and

distal tubular cells from rat kidney to chemically induced

toxicity Original Research Article

Toxicology, Volume 103, Issue 2, 30 November 1995, Pages 85-

103

Development of a primary mouse intestinal

epithelial cell monolayer culture system to

evaluate factors that modulate IgA transcytosis

Original Research Article

Mucosal Immunology 7, 818-828 (July 2014) |

doi:10.1038/mi.2013.98

Primary cell cultures of bovine colon epithelium: isolation

and cell culture of colonocytes Original Research Article

Toxicology in Vitro, Volume 14, Issue 5, October 2000, Pages

435-445

These monolayers

contained differentiated

epithelial cells that

displayed robust

transepithelial electrical

resistance.

WHY NEED

PRIMARY

CULTURE ??

2

4

3

1

Acquisition of tumor

Tumor dissociation techniques

Culture and Maintenance

Culture and Maintenance

An Organ-on-a-Chip, is a cell culture device that contains hollow channels

lined by living cells and tissues that mimic organ-level physiology.

The device is capable of producing levels of tissue and organ functionality not

possible with conventional culture systems and also allows real-time analysis

of biochemical, genetic, and metabolic activities within individual cells.

Researchers developed a bone

marrow-on-a-chip that reportedly

can reproduce the structure,

functions, and cellular makeup

of bone marrow

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