practical 4 07

Practical 4

In vitro cell cultivation

Tissue cultures

Cell cultures

In vitro cell cultivation

MAIN TYPES OF CELL CULTUREaccording to the cultivation period (and

survival in culture)

• primary cultures – derived from excised,

normal animal tissue

• continuous cultures – comprised of a single

cell type – cell lines

– normal diploid cells – maintain some degree of

differentiation, finite life (die after 30 divisions)

– established cell lines – tumour cells, transformed

cells – aneuploid number of chromosomes –

limitless availability (unlimited life span)

MAIN TYPES OF CELL CULTURE

• Animal cell culture– Mammalian cells

• Plant tissue culture– frequently used for

production of genetically modified plants

culture of macrophages

plant tissue culture -

callus

MAIN TYPES OF CELL CULTUREAccording to cell morphology

• epithelial cells– HeLa – tumour cell derived from the carcinoma of

human cervix – patient Helen Lacks

• fibroblasts– CHO – Chinese Hamster Ovary Cells– BHK – Syrian Hamster Kidney Cells

• lymphoblasts – HL60 – Human Leukemia – derived cells

• neuroblasts – SH-SY5Y – from human neuroblastoma

THE CELL ENVIRONMENT• temperature 37 ºC (incubator)• in some cases atmosphere in the incubator contains carbon dioxide

(approximately 5%)• sterile conditions (disinfection of the work surfaces, microbiological safety

cabinets)– (Main types of microbial contaminants – bacteria, fungi, Mycoplasma, viruses)

• culture medium– Basic Constituents of Media

• Inorganic salts• Trace elements• Buffering systems

– pH range should be 7,2 – 7,4

• Carbohydrates• Aminoacids• Vitamins• Proteins and peptides• Fatty acids and lipids• Serum – fetal bovine serum• Antibiotics

• cultivation flasks and tubes (plastic ware)

The biohazard box

log N

time

lag

log

stationary phase

decrease

Growth curve of the cell culture

Written test

• 10 minutes

• Don't forget to put down your name, your

group and the test version.

• In multiple choice questions more than 1

statement could be correct.

• Don't write anything on the question sheet!

Presentation

Cell cultures X laboratory animals

Tasks

Task 1INVERTED MICROSCOPE

• Inverted microscope is widely used for direct observation of cells in cultivation flasks.

• Observe various cell cultures using this microscope. Compare their morphology and density.

Task 2OBSERVATION OF HELA CELLS• Observe colonies of HeLa cells in the

small Petri dish. They were fixed with methanol and stained with the trypan blue solution.

• Before the observation remove the lid of the dish and observe colonies in the optical microscope using 10x magnification. Note the morphology of cells.

Task 3CELL QUANTIFICATION

• For the majority of manipulations using cell cultures, such as transfections, cell fusion techniques, cryopreservation and subculture routines it is necessary to quantify the number of cells prior to use. Using a consistent number of cells will maintain optimum growth and also help to standardise procedures using cell cultures.

• Estimate cell density using the Bürker haemocytometer. • Both sides of the chamber are filled with the cell suspension (2 x 10

microliters). View the cells under the light microscope using 20x magnification.

• Count the number of cells inside squares of the haemocytometer.• Calculate the cell density (CD) using equation• CD = n . 104 / s• CD – cell density (cells/millilitre)• n – number of cells• s – number of small squares

enumeration square

coverslip

Task 3: Cell quantification using hemocytometer

See microscopes on the table number 6.

Task 4 (see photo)CHROMOSOMES OF THE

CHO CELL LINE

• CHO is a continuous fibroblast cell line derived from the Chinese Hamster Ovary.

• Count chromosomes of the CHO cell in the photo.

• Describe the shape of chromosomes and explain your result.

Task 4 (see photos)• Count chromosomes of the CHO cell in the

photo.• Describe the shape of chromosomes and

explain your result

Results

Task 2: Observation of HeLa cells

Mitotic cells have circular shape.

Task 4: CHO cells

21 chromosomes

7

6

4

4

5

6

3

7

Aneuploid number of chromosomes is a consequence of cell transformation during the long time cultivation.

Karyotype of CHO cells

21 chromosomes

CHO cells have some features of tumor ones.

Karyotype of normal and tumor cell

normal male cell tumor cell

Rapid and uncontrolled proliferation of tumor cells leads to accumulation of numerical and structural chromosomal

abnormalities.

APPLICATION OF CELL CULTURES• clinical genetics

– prenatal examination (cultivation of amniocytes, chorionic villi or fetal blood lymphocytes)

– postnatal examination (cultivation of peripheral blood lymphocytes)

– chromosomal examination of tumors

• screening of mutagenicity (genotoxicity) and carcinogenicity of chemical compounds

• mapping of the human genome• cell differentiation studies (histology, embryology)• cell cycle and growth control studies (important for

oncology)• production of monoclonal antibodies• cloning of mammals (including man)• maintaining and isolation of viruses

Next practicals

• Chromosomal analysis (prenatal,

postnatal), karyotyping

• Test in previous topics (only practicals)

See you next week!

CHO cells