Cloning and Selection of Specific cell type

Clone; a population of cells are all descended from

a single parental cell

Cloning: A cell cultured from a single cell

Problems encountered during coning

1. Poor cloning efficiency

2. Survive for a limited number of generation

3. Heterogeneity may arise within the clone

as it is grow up for use

Methods:

1.Dilution cloning

seed cell at low density

incubate until colonies form

2.Feeder layer

render non proliferative cells by UV, x-ray or

drug treatment prior to cloning of test cells

3. Multiwell dish

one cell/well ……. Forming one clone

4. Semisolid media

5. Cloning in methocel over agar base

6. Selective media

ex. HAT for hybridoma

Improvements of plating efficiency

1. Hormones

ex. insulin, dexamethasone, synthetic

hydrocortisone

2. Intermediate metabolite

ex. pyruvate, -ketoglutarate, nucleosides

3. CO2

2% for HEPES( 20mM)

10% for DMEM….. hybridoma

4. Treatment of substrate

ex. polylysine treatment…..for human fibroblast

fibronectin

5.Trypsin

use purified trypsin

6. Multiwell dish

7. Semisolid media

ex. Hemopoietic stem cells or transformed

fibroblast

medium Agar

Prepare medium

Prepare cells

Cells count and resuspend in agar medium

Combine cells and agar medium

Cloning cells in suspension agar

cloning

Culture of Specific Cell Type

1.Epithelial cells

study models of stem cell differentiation

Responsible for organ function recognition

over growth of vascular endothelium

( improved by using serum free medium….)

Human Bronchial Epithelial Cells

Inhibition of fibroblast over growth

Methods Agent Tissue

Selective detachment trypsin fetal intestine,cardiac

muscle, epidermis

collagenase breast carcinoma

Selective detachment polyacrylamide various tumor

and substrate modification Teflon transformed cells

Collagen(pig skin) Epidermis

Confluent feeder layer Mouse 3T3 cells Epidermis

fetal human intestine breast

epithelium,normal

and malignant colon

carcinoma

Selective media D-valine kidney

MDCB170 colonic adenoma

phenonarbitone Liver

a. Epidermis Model for differentiation Idea tissue construct for organotypic culture Studies for sell interaction

Epidermal keratinocyte

( cultured by using 3T3 as a feeder layer)

keratinocyte foreskin

adjusting components of culture medium

( pH, temperature….)

add growth factor (EGF) or cholera toxin, or

isoprenaline

coculture mesanchyma cells with

keratinocyte

( improvement of keratinocyte differentiation)

Keratinocyte : corneal epithelial HCE-2

b. Breast using human intestine as a feeder layer culture with collagen gel( forms 3D structure

c. Gastrointestinal tract culturing colorectal carcinoma by using fetal human intestine as a feeder layer

d. Liver induce tyrosine aminotransferase of rat hepatoma by dexanmethasone( synthetic cortisone) as a study model of enzyme regulation culture liver cells on floating collagen sheet

e. Kidney prevent overgrowth of fibroblast by adding D- valine isolate tubule cells by collagenase

f. Bronchial and tracheal epithelia

culture by using floating collagen

treat with phorbal ester

use serum free medium with hydrocortisone,

insulin, transferring, estrogen and selenium

for carcinoma cells

2.Mesenchyma cell Derived from mesoderm

a. connective tissue( fibroblast cell)

may survive in simple medium

secret type I and type II collagen in medium

3T3 culture in high density may differentiate

into adipose tissue

b. muscle( skeletal, cardiac and smooth muscles) Skeletal, cardiac may be culture from chicken embryo Smooth muscle may be culture from blood vessel Use myosin or tropomyosin or calponin as a marker Use creatine phosphokinase as a indication of mature cells

c.cartilage( chondrocyte) may be cultured from chicken embryo somite, and stimulate by EGF

Human chondrocyte

d. Bone(osteoblast) treat by collagenase and EDTA

e.Endothelium

Culture from human umbilical cord or bovine aorta

and may be maintained by angiogenesis factor

Use factorVIII antigen or typeIV collagen as a

marker

Used for the study of angiogenesis in cancer cells

3.Haemopoietic cell

a.Normal haemopoietic cell

clone by agar or methocel

b.Normal and neoplastic leukocyte

using bone marrow culture as a feeder

layer for the culture of lymphoid, granulocyte

and erythroid stem cell

c. B and T cell lines

stimulate the growth of myeloid cell line by

using B and T cell growth factor

d.Human lymphobalstoid cell line

culturing blood cells in high cell density and

deep culture

e.Erythroid cell line erythroleukemia by infect mouse with mouse

RNA virus

http://www.jove.com/details.stp?id=2035Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

4. Neurorectodermal cell

a. Glia

grow only on treated substrate

culture from brain cells

use glial fibrillary acidic protein ( GFAP) as a

marker

b.Endocrine cell culture from adrenal or pituitary prevent fibroblast overgrowth by ethylmeriathio- salicyate

http://www.jove.com/details.php?id=2051Skeletal culture

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

http://www.jove.com/details.php?id=2389Primary motor neuron

Electrospinninga technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highlyaligned to completely random. In addition, fibers can be spun from a variety of materials, including biodegradable polymers such as poly-L-lacticacid (PLLA)

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Mouse Embryonic Lung Culture

http://www.jove.com/details.php?id=2316

Pulmonary Endothelial Cells provide a useful research model in many areas of vascular biology