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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
Cloning cells in suspension agar
medium
Prepare medium
Prepare cells
Cells count and
resuspend
in agar medium
Combine cells
and agar medium
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
Selective detachment
Agent
trypsin
Tissue
fetal intestine,cardiac
muscle, epidermis
collagenase
breast carcinoma
Selective detachment
polyacrylamide
various tumor
and substrate modification
Teflon
transformed cells
Collagen(pig skin)
Epidermis
Mouse 3T3 cells
Epidermis
fetal human intestine
breast
Confluent feeder layer
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 Dvaline
 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
Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice
http://www.jove.com/details.stp?id=2035
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 ethylmeriathiosalicyate
Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation
of Skeletal Muscle Stem Cells Skeletal culture
http://www.jove.com/details.php?id=2051
Primary motor neuron
http://www.jove.com/details.php?id=2389
Electrospinning
a technique for producing micro- to nano-scale fibers. Fibers can be electrospun
with varying degrees of alignment, from highly
aligned to completely random. In addition, fibers can be spun from a variety of
materials, including biodegradable polymers such as poly-L-lactic
acid (PLLA)
Isolation and Culture of Pulmonary Endothelial Cells from Neonatal
Mice
Pulmonary Endothelial Cells provide a useful research model in
many areas of vascular biology
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http://www.jove.com/details.php?id=2316