ANS/PLSS 433: Animal Cell Culture

	Animal tissue and/or cell culture has important implications
in biotechnology in the production of vaccines, pharmaceuticals, and 
antibodies (used in diagnostics), not to mention for basic research 
to understand the mechanisms of cell biology.  

I. Establishing cells in culture
	
	Three types of animal tissue culture:

	1. Organ culture.  The architecture characteristic of the 
		organ in vivo is retained, at least in part, in the 
		culture.  These tissues are generally grown on a 
		liquid/gas interface which favors retention of three 
		dimensional structure.

	2. Primary explant culture.  Fragments of tissues are grown 
		on a solid/liquid interface, where following 
		attachment, migration is promoted in the plane of 
		the solid substrate.  
 
	3. Cell culture.  Cells are dispersed (mechanically and/or 
		enzymatically) into a cell suspension which may then 
		be cultured as a monolayer on a solid substrate, or 	
		as a suspension in the culture medium--This type of 
		culture is called a primary culture.

II. Primary cell culture

	1. Dispersion enzymes--Proteolytic enzymes such as trypsin 
		or collagenase.
	2. Epithelial cells are usually the cell type of interest, 
		however, fibroblasts multiply the best--D-valine in 
		media will selectively promote growth of epithelial 
		cells.
	3. Primary cells can undergo a passage to secondary cultures 
		by detaching the cells with trypsin and/or EDTA, and 
		reseeding them in fresh media at high density.
	4. This can be done multiple times to form a cell line or 
		strain.  However, most primary cell lines have an 
		finite life, and will eventually fail to divide and 
		die.

III. Continuous cell lines

	1. Not all cells of primary cultures die.  Sometimes a few 
		cells in the culture will change in morphology and 
		growth characteristics, and they will not die--these 
		cells are transformed.
	2. Transformed cells are usually neoplastic--form cancer.
	3. Transformed cell lines can also be obtained by:
		a. Infecting cells with oncogenic viruses.
		b. Treating cells with carcinogenic chemicals.
		c. Propagating cells from tumors.
	4. Most transformed cells are aneuploid--possess extra 
		chromosomes.
	5. These cells are constantly changing; therefore stocks of 
		cells are generally maintained in liquid nitrogen.
	6. These cells may not have characteristics of normal cells 
		from which they originated.

IV. Growing Mammalian Cell Cultures

	1. Criteria for a culture media
		a. Must supply all nutritional requirements. 
		b. Must be buffered to maintain pH of 7.0-7.3.
		c. Must be isotonic with the cell cytoplasm.
		d. Must be sterile.
	2. Modes of cell growth
		a. Suspensions--Roller bottles and spinner flasks
		b. Monolayers--Culture dishes, T-flasks, Multiwell 
			plates.

V. Applications of Animal Cell Culture

	1. In Vitro Toxicity Testing
		a. Test chemical compounds on cells, rather than 
		whole animal.  Make animal welfare groups happy.
	2. Production of Viral Vaccines
		a. Early vaccines were made in whole animals.
		b. Now almost all are made in cell culture.
			i.	Live, avirulent (attenuated)
			ii.	Killed (inactivated)
		c. Major safety precautions
	3. Production of Pharmaceuticals
		a. Medically important proteins can be made in cell 
			culture.
		b. Problem with Post-translational modifications 
			that are seen when such proteins are produced 
			in bacteria aren't a problem.
		c. Most cultures are low yield, since large scale 
			culture of animal cells is difficult.
		d. Cell lines that would overproduce many of the 
			proteins of interest aren't available.  
			Cloning in animal cells should improve this 
			situation.  		
	4. Cloning to produce rare "complex" proteins for research 
		purposes or for Pharmaceuticals and Vaccines 
		(Chapter 5, Glick & Pasternak)
		a. Plasmids don't occur naturally in animals cells.
		b. Virus Vectors must be used.
			i.	Must be easily manipulated.
			ii.	Must contain unique restriction sites.
			iii.	Locations of restriction sites must be 
				known in respect to control sequences.
				--Beside replication, viruses need to 
					package the rDNA in viral coat 
					proteins.
				--Can cause size constraints on how much 
					foreign DNA you can clone into the 
					vector.
			iv.	Need selectable markers.
			v.	Commonly viral vectors include SV40, 
				Adenovirus, Vaccinia, Bovine Papillomavirus 
				(BPV),Human Papova BK Virus (BKV), 
				Polyomavirus, and Retrovirus.
		c. The introduction of unpackaged DNA into animal cells is 
			called transfection.
			i.	Most common method is co-precipitation of 
				DNA and calcium phosphate which is 
				phagocytosed into the cell.
			ii.	Other methods include DEAE-dextran, 
				Polybrene, Protoplast fusion, 
				Lithium acetate, Electroporation, 
				Liposomes, and 	Microinjection.
 	
	5. Other methods to produce proteins (Chapter 5, Glick & Pasternak)

		A. Yeast--S. Cervisiae
			a. Old system, still used
			b. Why used.
				1. Single celled, easily grown
				2. Strong Promoters, Natural plasmid
						--2 µm plasmid
				3. Does many post-translational modifications
				4. Releases few proteins, so engineered 
					proteins can be easily purified.
				5. FDA safe--already used in baking and 
					brewing
			c. Proteins made
				1. Vaccines (Hepatitis B, Malaria)
				2. Diagnostics (Hepatitis C, HIV)
				3. Therapeutics (Insulin, Growth factors, 
					Clotting factors)
		B. Insect system-- Baculovirus
			a. Has become a popular method
			b. Autographa californica multiple nuclear 
				polyhedrosis virus
					--alfalfa looper
			c. Uses E.coli base vector with AcMNPV
			d. Co-transfect vector and virus DNA
			e. Crossover occurs
			f. Protein found in occlusion bodies

	6.  Scientific Research

		A.	Understand mechanisms of action/treatments
			a.	Tissues from Organs
			b.	Cancers
		B.	Donor for Nuclear Transfer (Cloning)

	7.  Production of Antibodies