Dr: RAWIA BADR Associate Professor of Microbiology&Immunology

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2 Dr: RAWIA BADR Associate Professor of Microbiology&Immunology

3 Cell culture Commonly refers to the culture of animal cells and tissues, while the more specific term plant tissue.culture is used only for plants In cell culture, the cells are capable of dividing, increasing in size and in a batch culture, single cells are allowed to act as independent units, such as a bacterium or fungus until limited by some culture.variables such as nutrient depletion

4 .Cell culture-cont When the process is carried out in the laboratory, it is in glass as called Cell Culture, It occurs in vitro. in life opposed to occur in vivo Tissue culture is often used interchangeably with cell.culture

5 History The history of cell culture dates back to early 20 th century, when the first development of cell culture was to study, under the microscope, normal physiological events such as nerve development. Animal cell culture didn t become routine laboratory technique untill the 1950s, where the need for cell culture, especially at large scales, became apparent with the need for viral vaccines when major epidemics of polio in the 1940s promoted a lot of efforts to develop an effective vaccine.

6 Cell culture-types Freshly isolated cultures from mammalian tissues are known as primary cultures until subcultured. At this stage cells are usually heterogeneous but still closely represent the parent cell types as well as in the expression of tissue specific properties.

7 Types-cont. After several subcultures onto a fresh media, the cell line will either die out or transform to become a continuous cell line. Such cell lines show many alterations from the primary cultures including morphological chromosomal variations. changes and

8 Cell Culture-Forms Cells can be grown either in suspension or adherent.cultures Some cells naturally live in suspension, without being attached to a surface, such as cells found in the blood stream. There are also cell lines that have been modified to be able to survive in suspension cultures so that they can be grown to a higher density than.adherent conditions would allow

9 Forms-cont. Adherent cells (organs) require a surface, such as tissue culture plastic, which may be coated with extracellular matrix components to increase adhesion properties and to provide other signals needed for process of. growth and differentiation

10 Cell Culture-Harvesting When to harvest cells Cells are harvested when the cells have reached a population density which suppresses growth. Ideally, cells are harvested when they are in a semi-confluent state and are still in the log phase. Cells that are not passaged and are allowed to grow to a confluent state can sometime lag for a long period and some times may never recover.

11 This picture shows the uninfected Vero cell line at 48 h after seeding of the culture. Several nucleoli are visible inside each nucleus. Cells have been stained with hematoxylin and eosin.

12 This picture shows uninfected Vero cell line. Complete monolayer sheet h after seeding of the culture.

13 Cell Culture-Applications Investigation of the normal physiology or biochemistry of cells e.g. studies of cell metabolism. Testing the efficacy of various chemical compounds or drugs on specific cell types (normal or cancerous cells). Studying the sequential or parallel combination of various cell types to generate artificial tissues e.g. artificial skin. The possibility of generating artificial tissues is known as tissue engineering.

14 Applications-cont. Production of Biological substances by recombinant DNA ( rdna) technology in cell cultures include enzymes, synthetic hormones, immunobiologicals (monoclonal antibodies, interleukins, lymphokines and anticancer agents) and for studying oncogenesis.

15 Applications-cont. Production of hybrid cells (hybridoma) from the fusion of two or more cells capable of continuous production of a single type of antibody. These antibodies have diagnostic and therapeutic value and are now produced commercially.

16 Applications-cont. Expression of mammalian genes in bacteria, although many simpler proteins can be produced using rdna in bacterial cultures, the more complex proteins that are glycosylated ( carbohydrate - modified ) could not be produced in bacteria, as they do not have the appropriate metabolism to add sugar chains to these proteins. These must be made in cell culture e.g. the hormone erythropoietin.

17 Applications-cont. Production of vaccines e.g. polio, measles, mumps, rubella and chickenpox are currently made in cell cultures. pandemic threat, 1 N5Nowadays due to the H research into using cell culture for influenza vaccines. is going ahead Recombinant DNA-based vaccines, such as that made using human adenovirus as a vector or. as adjuvants

18 Cell Culture-advantage The major advantage of using cell culture for any of these applications is: the consistency and the reproducibility of results obtained.

19 Cell Culture-disadvantage The disadvantage is that after a period of continuous growth when cells continue to divide.and to grow filling the available area or volume Several obstacles are aroused

20 Disadvantage-cont. Nutrient depletion in the growth media. Accumulation of apoptotic/necrotic (dead) cells. Cell-to-cell contact can stimulate cell cycle arrest, causing cells to stop dividing contact inhibition. Cell-to-cell contact can stimulate cellular differentiation.

21 Disadvantage-cont. Cells characteristics can change, and may become quite different from those found in the starting population. Cells can adapt to different culture environments (e.g. different nutrients, temperatures and salt concentration ) by varying the activities of their enzymes leading to mutants formation.

22 Others-Plant cell culture methods Plant cells have been cultured to produce many ingredients needed by the food industries. Much progress has been made in recent years in understanding the basics of plant metabolism. A wide range of food ingredients including flavors, essential oils, sweeteners and.antioxidants have been produced in culture

23 Bacterial/Yeast cell culture methods For bacteria and yeast, small quantities of cells are usually grown on a solid support that contains nutrients embedded in it ( agar), while large-scale cultures are grown with the cells suspended in a.nutrient broth

24 Viral cell culture methods The culture of viruses requires the culture of cells of mammalian, plant, fungal or bacterial origin as hosts.for the growth and replication of the virus Whole wild type viruses, recombinant viruses or viral products may be generated in cell types other than their natural hosts under suitable conditions. Depending on the species, the infection and the replication, viral infections result in host cell lysis and formation of a.viral plaque

25 These pictures show CPE typical of herpes simplex virus onvero cells. The small syncytia,or multinucleated giant cells, result from fusion of cell membranes bearing viral glycoproteins. Alterations in the cell nuclei and cytoplasmic tails between the cells are seen. The cell cultures have been stained with hematoxylin and eosin. 72 h after virus inoculation.

26 Uninfected monolayer sheet of Vero cells. Phase contrast microscopy 40.

27 Inoculation with a clinical specimen. Typical cytopathic effect (CPE) caused by HSV-1. Phase contrast microscopy 200.

28 Shell vial culture positive for HSV-1. Brightly fluorescing (apple green) HSV-1 infected cells. Indirect immunofluorescence assay, x 250

29 Shell vial culture positive for HSV-1. HSV-1 infected cells (stained dark brown). Uninfected cells appear bluish purple due to counterstaining with H&E Indirect immunoperoxidase assay, 500.

30 Tube culture positive for HSV-1. HSV-1 infected cells (stained dark brown). Uninfected cells appear bluish purple due to counterstaining with haematoxylin. Indirect immunoperoxidase assay.

31 Thinking-ape

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