Animal Cell Culture

Prepared byAmit ShresthaM- pharm(Pharmacology)Mallige College Of Pharmacy

Institute of Animal Health and Veterinary Biologicals

2

History• History• The 19th-century English physiologist Sydney Ringer developed salt solutions containing

the chlorides of sodium, potassium, calcium and magnesium suitable for maintaining the beating of an isolated animal heart outside of the body.[1] In 1885, Wilhelm Roux removed a portion of the medullary plate of an embryonic chicken and maintained it in a warm saline solution for several days, establishing the principle of tissue culture.[3] Ross Granville Harrison, working at Johns Hopkins Medical School and then at Yale University, published results of his experiments from 1907 to 1910, establishing the methodology of tissue culture.[4]

• Cell culture techniques were advanced significantly in the 1940s and 1950s to support research in virology. Growing viruses in cell cultures allowed preparation of purified viruses for the manufacture of vaccines. The injectable polio vaccine developed by Jonas Salk was one of the first products mass-produced using cell culture techniques. This vaccine was made possible by the cell culture research of John Franklin Enders, Thomas Huckle Weller, and Frederick Chapman Robbins, who were awarded a Nobel Prize for their discovery of a method of growing the virus in monkey kidney cell cultures.

3Note:-The neural plate is a key developmental structure that serves as the basis for the nervous system

Introduction:• Cell culture is the complex process by which cells are grown under

controlled conditions, generally outside of their natural environment. In practice, the term "cell culture" now refers to the culturing of cells derived from multi-cellular eukaryotes, especially animal cells. However, there are also cultures ofplants, fungi and microbes, including viruses, bacteria and protists. The historical development and methods of cell culture are closely interrelated to those of tissue culture and organ culture.

4

5

Purpose

Cell culture is essential for growth of  viruses because they are obligate intracellular parasites; they cannot replicate in any cell-free medium, and they require living cells from a suitable host within which to multiply.  Animals such as mice and embryonated avian eggs may be used for the propagation of viruses, but for various reasons (time, cost, uniformity, ease of handling, animal welfare, etc.), the propagation of most viruses in cultured living cells is the method of choice today.  Cell culture is also used in recombinant DNA technology and many in vitro assays. 

Isolation

• Cells can be isolated from tissues for ex vivo culture in several ways. Cells can be easily purified from blood; however, only the white cells are capable of growth in culture. Mononuclear cells can be released from soft tissues by enzymatic digestion with enzymes such as collagenase, trypsin, or pronase, which break down the extracellular matrix. Alternatively, pieces of tissue can be placed in growth media, and the cells that grow out are available for culture. This method is known as explant culture.

Collagenase Collagenases are enzymes that break the peptide bonds in collagen.

Pronase is a commercially available mixture of proteinases isolated from the extracellular fluid of Streptomyces griseus. Activity extends to both denatured and native proteins leading to complete or nearly complete digestion into individual amino acids.

tripsin  is a serine protease found in the digestive system of many vertebrates, where it hydrolyses proteins.

6

7

Cell Cultures/ Dispersion Cultures

• Types

• Primary cell cultures

• Diploid cell cultures

• Continuous cell lines

8

Primary cell cultures.  

When cells are taken freshly from animal tissue and placed in culture, the cultures consist of a wide variety of cell types, most of which are capable of very limited growth in vitro, usually fewer than ten divisions. These cells retain their diploid karyotype, i.e., they have the chromosome number and morphology of their tissues of origin. They also retain some of the differentiated characteristics that they possessed in vivo. Because of this, these cells support the replication of a wide range of viruses.  Primary cultures derived from monkey kidneys, mouse fetuses, and chick embryos are commonly used for diagnostic purposes and laboratory experiments.

a cell line derived directly from the parent tissue. Cells in primary culture have the same karyotype and chromosome number as those in the original tissue.Can be subculture only one or twice e.g-primary monkey, baboon kidney

9

2. Diploid cell strains.  

Which are derived from human fetal tissue and can be subcultured 20-50 times e.g-human diploid fibroblast such as MRC-5(human fetal lung fibroblast establish from normal lung tissue of 14 week old male foetus)

Some primary cells can be passed through secondary and several subsequent subcultures while retaining their original morphological characteristics and karyotype. Subcultures will have fewer cell types than primary cultures.  After 20 to 50 passages in vitro, these diploid cell strains usually undergo a crisis in which their growth rate slows and they eventually die out.  Diploid strains of fibroblasts derived from human fetal tissue are widely used in diagnostic virology and vaccine production.

3. Continuous cell lines.  Derived from tumors of human or animals e.g- Vero, Hep2

Certain cultured cells, notably mouse fetal fibroblasts, kidney cells from various mammalian species, and human carcinoma cells, are able to survive the growth crisis and undergo indefinite propagation in vitro.  After several passages, the growth rate of the culture slows down; then isolated colonies of cells begin to grow more rapidly than diploid cells, their karyotype becomes abnormal (aneuploid), their morphology changes, and other poorly understood changes take place that make the cells immortal. The cells are now "dedifferentiated," having lost the specialized morphology and biochemical abilities they possessed as differentiated cells in vivo. Continuous cell lines such as KB and HeLa, both derived from human carcinomas, support the growth of a number of viruses.  These lines and others derived from monkey kidneys (e.g., Vero), mouse fetuses (L929), and hamster kidneys (BHK) are widely used in diagnostic and experimental virology.  Continuous cell lines have been established from many types of vertebrate and invertebrate animal tissues and are available from the American Type Culture Collection. 

11

Primary Cell Culture

• Sterilization of Cubicle

• Preparation of Media• Preparation of

dispersion solution• Preparation of

Monolayers

12

Sterilisation of Cubicle (compartment)

• Fumigation• 35 ml of formaline and 17.5 g of potassium

permanganate per 100 cubic feet of air space

• Keep the cubicle closed for 65 hours

• Working Surface Sterilization• Mop with 5% phenyl/ 1/40 solution of dettol

• Use 1 % Copper sulphate for fungal disinfection

13

Cell Culture Media

• Balanced Salt Solution (BSS)

• Glucose

• Amino acids

• Vitamins

• Sodium bicarbonate

• Phenol red

• Fetal Calf Serum

14

Culture Media Prepare stock antibiotic solution

Weigh the required amount of Ready made Balanced Salt Medium and

add sterile water

Add the required amount of antibiotic stock solution into BSS

Adjust the pH of the solution using 2.8% / 7.5% Sodium bicarbonate

or 0.1 N Hydrochloric acid to 7.4

Sterilize using 0.2 Seitz filter applying positive pressure.

Add sterile heat inactivated fetal calf serum to make a final

concentration of 15%

Store at 4o C.

15

Dispersion solution

• Weigh Calcium Magnesium free Buffer premix and add sterile water

• Add 0.1 % w/v Trypsin powder

• Adjust pH to 7.4 to 7.6

• Sterilize by Filtration through Seitz filter

• Store at - 20o C

Primary Cell Culture

Chicken Embryo Fibroblast

17

Materials Ten to Eleven days Embryonated Chicken Eggs

Forceps, Scissors, Rubber bulbs

Pipettes, Beakers, Centrifuge tubes, Flasks, Petri dishes, Trypsinisation flask

Seitz filter assembly, Vacuum/air pump

Mono-pan analytical balance

pH meter

Magnetic stirrer, Teflon coated magnetic stirrer, Cyclo-mixer

Refrigerated Centrifuge

Laminar Air Flow Apparatus

18

Preparation of Chicken Embryo Fibroblast Preparation of Chicken Embryo Fibroblast MonolayerMonolayer

1. Sterilize Outer shell of the Eggs 2. Remove the Shell, Shell membrane and CAM3. Wash embryo with BSS4. Remove Head, Limbs and Internal Organs5. Wash several times with BSS6. Cut into small pieces of ~ 1mm thickness7. Wash in BSS8. Trypsinize /.9. Filter with a cheese cloth10. Centrifuge and pack cells11. Resuspend in growth medium12. Repeat step 10 and 11, two more times13. Count the cells and adjust to a concentration of 10 6 cells per ml14. Seed Culture tubes/ flasks/ Bottles and Incubate at 37 o C for 48 to 72

hours.

19

procedure

• Disinfect the surface of the egg over the air sac.  With scissors or the blunt-end of a forceps, break the shell over the air sac

• Opening of egg through a circular incision around the airsac

•10- to 12-day-old embryonated eggs

20

• Transfer the live embryo to a petri dish containing media

21

• Collect more embryos

22

• Remove the appendages and viscera

23

• Collect the remaining portion

24

• Cut the embryo in to small parts (about 1 mm)

25

• Trypsinise in a flask

Add 10 ml of sterile warm trypsin solution to fragments .

26

• Keep in magnetic stirrer for about 30 minutes

27

• Centrifuge the cell suspension

28

29

• Wash the cells 3 more times.

30

• Mix well in cyclo mixer

31

• Incubate for about 30 min at 4oc

32

• Dispense in cell culture bottles

33

• Incubate at 37oc in a CO2 incubator

34

• Preservation of Cultured Cells by Freezing 

• 50 to 80% of the cells of a healthy culture will survive freezing.

• .  Cells must be stored at -70oC or lower

36

Ex vivo (Latin: out of the living) means that which takes place outside an organism. In science, ex vivo refers to experimentation or measurements done in or on living tissue in an artificial environment outside the organism with the minimum alteration of the natural conditions. The most common "ex vivo" procedures involve living cells or tissues taken from an organism and cultured in a laboratory apparatus, usually under sterile conditions and no alterations done for a few hours up to 24 hrs. Experiments lasting longer than this using living cells or tissue are typically considered to be "in vitro". Ex-vivo conditions allow experimentation under highly controlled conditions impossible in the intact organism, albeit at the expense of looking at the tissue in its "natural" environment. One widely performed ex vivo study is the chick chorioallantoic membrane (CAM) assay. In this assay, angiogenesis is promoted on the CAM membrane of a chick embryo outside the organism (chicken). Ex vivo studies are usually performed in vitro, although the use of these two terms is not synonymous.

In vitro (Latin for within the glass) refers to the technique of performing a given procedure in a controlled environment outside of a living organism. Some may argue that in vitro refers to a process that is created in a "test tube"; however, Robert Kail and John Cavanaugh on page 58 in the 4th edition of Human Development: A Life-Span View cite that in fact the process is contained in a petridish. Many experiments in cellular biology are conducted outside of organisms or cells; because the test conditions may not correspond to the conditions inside of the organism, this may lead to results that do not correspond to the situation that arises in a living organism. Consequently, such experimental results are often annotated with in vitro, in contradistinction with in vivo.

What's the difference between ex vivo and in vitro?Or is it the same thing

Institute of Animal Health and Veterinary Biologicals

37

karyotypethe chromosomal constitution of the cell nucleus; by extension, the photomicrograph of chromosomes arranged

38

Diploid cultures vs. continuous cell linesWhat is the difference between a diploid culture and a continuous cell line?-Diploid cultures have a finite lifespan. They can undergo a maximum of 20-80 PDLs (Population Doubling Level) before they senesce. Normal human cells, such as human skin fibroblasts, are one example of diploid cells.-Continuous cell lines are immortalized cell lines with an infinite lifespan. These usually either come from tumor tissue or have been deliberately immortalized or transformed. However, many rodent cell lines spontaneously transform.

39

Formulations of Media and Solutions 

          Saline A

Ingredient                g/l

 NaCl                        8.0                    

 KCl                          0.4                

 NaHCO3                   0.35                

 Glucose                    1.0             

 Phenol red                0.05            

Add distilled H2O to 1 liter.   Filter sterilize.  Saline A is usually prepared as a 10X solution and stored at -20oC. 

     Saline-Trypsin-EDTA (STE) 

Ingredient                                  g/l

 Trypsin (1:250)                         0.5 

 EDTA (disodium salt)                  0.2  

 NaCl                                       8.0 

 KCl                                         0.4  

 NaHCO3                                 0.35  

 Glucose                                   1.0

 Phenol red                              0.05 

Add distilled H2O to 1 liter.   Dissolve trypsin first by slowly stirring in ~200 ml H2O (avoid denaturation).  Add remaining ingredients and filter sterilize.  STE is usually prepared as a 10X solution and stored at -20oC.