cloning and selection of specific cell type clone; a population of cells are all descended from a...
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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
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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
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Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice
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