1. 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

2. 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

3.  Improvements of plating efficiency 1. Hormones ex. insulin, dexamethasone, synthetic hydrocortisone 2. Intermediate metabolite ex. pyruvate,  -ketoglutarate, nucleosides 3. CO 2 2% for HEPES( 20mM) 10% for DMEM….. hybridoma 4. Treatment of substrate ex. polylysine treatment…..for human fibroblast fibronectin

4. 5.Trypsin use purified trypsin 6. Multiwell dish 7. Semisolid media ex. Hemopoietic stem cells or transformed fibroblast

5. mediumAgar Prepare medium Prepare cells Cells count and resuspend in agar medium Combine cells and agar medium Cloning cells in suspension agar

6. cloning

7. 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….)

8. Human Bronchial Epithelial Cells

9. 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

10. 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)

11. Keratinocyte : corneal epithelial HCE-2

12. 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

13. 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

14. 2.Mesenchyma cell Derived from mesoderm

15. 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

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

17. 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

18. 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

19. 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=2035 Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

20. 4. Neurorectodermal cell a. Glia  grow only on treated substrate  culture from brain cells  use glial fibrillary acidic protein ( GFAP) as a marker

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

22. http://www.jove.com/details.php?id=2051 Skeletal culture Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

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

24. 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