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PTC - C7 (Basic Animal Cell Culture) Cheat Sheet (DRAFT) by

Brief summary of Chapter 7 (Basic Animal Cell Culture) of Plant and Tissue Culture Subject

This is a draft cheat sheet. It is a work in progress and is not finished yet.

Overview

Applic­ation

1. The study of basic cell biology, cell cycle mechan­isms, specia­lized cell function, cell–cell and cell–m­atrix intera­ctions.
2. Toxicity testing to study the effects of new drugs.
3. Gene therapy for replacing nonfun­ctional genes with functional gene-c­arrying cells.
4. The charac­ter­ization of cancer cells, the role of various chemicals, viruses, and radiation in cancer cells.
5. Production of vaccines, mABs, and pharma­ceu­tical drugs.
6. Production of viruses for use in vaccine production (e.g., chicken pox, polio, rabies, hepatitis B, and measles).

Types of Tissue Culture

Explant Culture Procedure

1. Obtaining the Explant
-obtained surgically using sterile equipment from mammals, rodents or avian organs or tissues
 
-ex 1: a piece of gingival tissue following tooth extraction can be removed as an explant to establish human gingival fibrob­lasts
 
-ex 2: a piece of adipose tissue can be used to establish mesenc­hymal stem cells
2. Cut and Clean the Explant
-place the explant in a petri dish containing around 1-2 mL of incomplete medium (medium without serum)
 
-using a sharp surgical blade, you can cut it (usually around 1×1 mm pieces)
 
-collect the pieces of explant using a sterile forceps and wash gently
 
-washing can be done by transf­erring pieces into a centrifuge tube containing around 0.5 mL of incomplete medium
 
-gently mix by pipetting the medium 4 to 5 times, and allow the pieces to settle down and remove the upper medium
 
-can be repeated 2 or 3 times
3. Culturing the Explants
-obtained explants are asepti­cally placed on a coated surface and allowed to attach to the surface in the presence of a rich culture medium
 
- medium ex: basal minimal media, Dulbecco’s Modified Eagle Medium (DMEM) or Minimum Essential Medium Eagle (MEM) supple­mented with 10-15% serum
 
-cultured in standard tissue culture conditions (pH 7.2-7.4, temper­ature 37°C, 5% CO2 and humidity) to allow for cell migration and prolif­eration
 
-change the media every 3 days without disturbing the explants
 
-depending upon the health and age of the tissue, cells emerge out of the explant within 15-30 days
 
-once outgrowth of cells starts from the explant, add 5 mL of medium to the flask in subsequent days
4. Once outgrowth of cells starts from the explant, add 5 mL of medium to the flask in subsequent days
-after the explants are completely surrounded by the cells, you can trypsinise the cells and subcul­ture.
 
-it is better to use a lower concen­tration of trypsin (e.g. <0.25% of trypsin for 5 min)
 
-choose an approp­riate size of flask for seeding, depending on the total number of cells obtained

Pros and Cons of Types of Tissue Culture

Primary Culture

-cultures prepared from tissues taken directly from animals
1. Organ Culture
(googl­e)-­organ culture is able to accurately model functions of an organ in various states and conditions by the use of the actual in vitro organ itself
 
-maint­enance of a piece of tissue, a part of organ or a whole organ in vitro
2. Primary Cell Culture
-when taken tissue is dissoc­iated, mechan­ically or enzyma­tic­ally, into single cells which could be plated on a coated surface
3. Slice Tissue Culture
-referred to as explant or organo­typic cultures
 
-small pieces of tissue of interest are simply allowed to attach to an approp­riate substrate and are cultured in enriched media
4. Re-agg­regate Culture
-disso­ciated cells is kept in suspension rather than allowed to settle on and attach to solid substrate
 
-cells tend to re-agg­regate into small balls
 
-allowed cells cells to develop in three dimensions
5. Histotypic or histoc­ulture
-culture of intact tissues
(Google) - Histotypic culture is defined as three-­dim­ens­ional culture of one cell type, while the term organo­typic implies the intera­ction of two or more cell types from a complex tissue or organ.

Types of Cells

1. Epithe­lia­l-Like
-cells that are attached to a substrate and appear flattened and polygonal in shape
2. Lympho­bla­st-Like
-cells that do not at­tach normally to a substrate but re­main in suspension with a spherical shape
3. Fibrob­las­t-Like
-cells that are attached to a substrate and appear elongated and bipolar, frequently forming swirls in heavy cultures
It is important to remember that the cul­ture conditions play an important role in determ­ining shape and that many cell cul­tures are capable of exhibiting multiple morpho­logies.

Types of Cell Culture

1. Primary Cell Culture
-Adherent Cell Culture
 
-Suspe­nsion Cell Culture
2. Secondary Cell Culture
-
3. Cell Line
-Finite Cell Line
 
-Conti­nuous Cell Line

1. Primary Cell Culture

-maint­enance of growth of cells in culture medium using suitable glass or plastic containers
-using the mechanical or enzymatic methods
-disso­ciated directly from the parental tissue (such as kidney, liver)
-they will attach, divide and grow
 
2 types of primary cell culture depending upon the kind of cells in culture
a) Anchorage Dependent /Adherent cells
-require attachment for cell growth
 
-monolayer culture system
 
-usually derived from tissues of organs such as kidney where they are immobile and embedded in connective tissue
 
(googl­e)-have to be detached from surface before being subcul­tured
 
(googl­e)-­growth limited to surface area
b) Suspension Cultur­e/A­nch­orage Indepe­ndent cells
-do not require attachment for cell growth/do not attach to the surface of the culture vessels
 
-all suspension cultures are derived from cells of the blood system because these cells are also suspended in plasma in vitro e.g. lympho­cytes

Pros and Cons of Primary Cell Culture

2. Secondary Cell Cultures

-When a primary culture is sub-cu­ltured, it becomes known as secondary culture or cell line.

3. Cell Line

-cell population derived from a primary culture at the first subculture
(googl­e)-­usually clonal, meaning that the entire population originated from a single common ancestor cell
-the term does not imply homoge­neity or the degree to which a culture has been charac­terized
 
may be finite or continuous depending upon whether it has limited culture life span or it is immortal in culture
a) Finite Cell Lines
-cell lines which have a limited life span and go through a limited number of cell genera­tions
 
-growth rate is slow and doubling time is around 24-96 hours
b) Continuous Cell Lines
-grow indefi­nitely
 
-cell lines transf­ormed under laboratory conditions or in vitro culture conditions give rise to continuous cell lines
 
-growth rate is rapid and doubling time is 12-24 hours
c) Transf­ormed Cell Line
-cell lines obtained from tumor cells
d) Clonal Cell Line
-cells could be cloned in continuous cell lines to obtain geneti­cally homogenous population

Pros and Cons of Finite Cell Lines

Pros and Cons of Continuous Cell Line

Difference of Normal and Transf­ormed Cells

Normal Cells
Transf­ormed Cells
1. Anchor­age­-de­pendent (except blood cells)
1. Nonanc­hor­age­-de­pendent
2. Densit­y-d­epe­ndent inhibition of prolif­eration
2. No densit­y-d­epe­ndent inhibition of prolif­eration
3. Mortal; Finite Cell Line
3. Immortal; Continuous Cell Line
4. Contact Inhibi­tion; Monolayer Culture
4. No Contact Inhibi­tion; Multilayer Culture
5. Dependent on external growth factor signals for prolif­eration
5. May not need an external source of growth factors
6. Greater retention of differ­ent­iated cellular function
6. Typically loss of differ­ent­iated cellular function
 
-shorter population doubling time
 
-reduced substrate adhesion
 
-genetic instab­ility (e.g. show hetero­ploidy and aneupl­oidy)

Contac­t-I­nhi­bition of Growth

Densit­y-d­epe­ndent Inhibition of Prolif­eration

-reduction in prolif­erative activity that correlates with the attainment of conflu­ency, that is，occ­upancy of all available attachment surface
-can occur before confluence is reached, and reflects diminished nutrient supply and the release of cell-d­erived factors (including waste products) into the medium
 
Saturation Density
-popul­ation density (cells­/cm2) at the point when it reaches densit­y-d­epe­ndent inhibition of growth
 
-popul­ation density (cells­/cm2) at the point when it reaches densit­y-d­epe­ndent inhibition of growth

Cell Ageing in Culture

-also known as In vitro cell senescence
-involve progre­ssive altera­tions in a number of cell charac­ter­istics
 
Normal cell lines commonly have a finite lifespan, that is, they do not grow beyond a finite number of cell genera­tions (popul­ation doubli­ngs).
-Eg, the lifespan of normal diploid fibrob­lasts is in the range of 50-70 population doubling.
 

Transf­ormed Cells

-cancerous cells
-possess all six hallmarks of cancerous cells :
1. Growth factor indepe­ndency
2. No response to growth inhibitors
3. Evasion of apoptosis (Natural cell death)
4. Can promote angiog­enesis (the develo­pment of new blood vessels)
5. Unlimited prolif­eration - rapid increase
6. Invasive - tending to spread very quickly and undesi­rably or harmfully

Immort­ali­zation

-Cell lines that have unlimited lifespan arc termed immortal or, prefer­ably, continuous
 
the term immort­alized and transf­ormed are not synonymous
Although infinite lifespan is generally considered to be a charac­ter­istic of transf­ormed cells, not all continuous cell lines exhibit altera­tions in growth control attributed to cellular transf­orm­ation.
 
Immort­alized Cells
-not yet cancerous, but have sufficient mutations to be able to be passaged forever, unlike non-tr­ans­formed, non-im­mor­talized cells, which all have a finite passage number
-popul­ation of cells from a multic­ellular organism due to mutation, which can escape normal cellular senescence and keep undergoing division
-this kind of cells can grow in vitro for prolonged periods

Cell Strain

-describe a subcul­tured population selected on the basis of its expression of specific proper­ties, functional charac­ter­istics, or markers

Clonal Culture / Clonal Selection

-clone
-estab­lis­hment of a cultured cell population derived from a single cell

Sub-cu­lturing (or passage)

-Transfer or transplant cells of an ongoing culture to a new culture vessel so as to propagate the cell population or set up replicate cultures for study.
-Subcu­lturing or splitting cells is required to period­ically provide fresh nutrients and growing space for contin­uously growing cell lines.
-Such cultures may be called secondary cultures (first subculture from primary culture)
 
Criteria for Subcul­turing
1. Cell concen­tra­tion: should not exceed 1 x 10^6 cells/mL for most suspen­sio­n-g­rowing cells
2. pH: which is linked to cell concen­tra­tion, and declines as the cell concen­tration rises
3. Time since last subcul­ture: should fit a regular schedule
4. Cell production requir­ements: for experi­mental or production purposes

Pros and Cons of Animal Cell Culture

Advantage
Disadv­antage
1. Controlled physio­che­mical enviro­nment (pH, temper­ature, osmotic pressure, O2, osmolarity etc.)
1. Expertise is needed, so that behavior of cells in culture can be interp­reted and regulated.
2. Controlled and defined physio­logical con­di­tions - nutrient concen­tra­tion, cell to cell intera­ctions, hormonal control.
2. Need of expertise and technical skill for the develo­pment, and regular use of tissue culture.
3. Homoge­neity of cell types (achieved through serial passages)/ Homogenous genetic population
3. Ten times more expensive for same quan­tity of animal tissue; therefore, reasons for its use should be compel­ling.
4. Econom­ical, since smaller quantities of reagents are needed than in vivo.
4. Unstable aneuploid chromosome consti­tu­­tion.
5. Legal, moral and ethical questions of ani­mal experi­men­tation are avoided.
5. Cost factor is a major limita­tion.
6. Cost effective screening assays
-Estab­lis­hment of infras­tru­cture, equipment and other facilities are expensive.
7. Easy production of biopha­rma­ceu­ticals
-It is estimated that the cost of production of cells is about 10 times higher than direct use of animal tissues.
8. Available in adequate numbers to do chemical study
6. Control of the enviro­nmental factors (pH, temper­ature, dissolved gases, disposal of biohaz­ards) is not easy.
9. Easy to add genes (trans­fec­tion) or regulate protein levels (RNAi)
7. The native in vivo cells exist in a three- dimens­ional geometry while in in vitro tissue culture, the propag­ation of cells occurs on a two dimens­ional substrate.
 
-Due to this, the cell to cell intera­ctive characters are lost.
 
8. The cell lines may represent one or two types of cells from the native tissue while others may go unrepr­ese­nted.
 
9. Tissue culture techniques are associated with the differ­ent­iation i.e. loss of the characters of the tissue cells from which they were originally isolated.
 
-This happens due to adaptation and selection processes while culturing.
 
10. Continuous cell lines may result in genetic instab­ility of the cells.
 
-This may ultimately lead to hetero­geneity of cells.

Growth Measuring Methods

1. Direct Methods
2. Cells
-Packed Cell Volume
 
-Cell count and viability
 
-Colony forming unit
 
-Optical density (OD)
3. Tissues
-Fresh weight and dry weight
4. Indirect Method
-Mostly used for large-­scale cultures

Growth Observing

1. Increase in turbidity of cells
2. Increase in size of tissues/ explants
-swelling
 
-curling
 
-proli­fer­ation
3. Decrease in turbidity and size
-death
 
-apoptosis and necrosis
4. Micros­copic observ­ation
-Stere­oscope
 
-Inverted microscope
 
Necrosis is caused by factors external to the cell or tissue, such as infection.

Charac­ter­ization of Cell Lines

a) growth rate
b) karyot­yping (C11)

Growth Curve

-estab­lished taking into consid­eration the population doubling time, a lag time, and a saturation density of a particular cell line.
1. Lag Phase
The time the cell population takes to recover from such sub culture, attach to the culture vessel and spread.
2. Log Phase
In this phase the cell number begins to increase expone­nti­ally.
3. Plateau Phase
During this phase, the growth rate slows or stops due to exhaustion of growth medium or conflu­ency.

Bacterial Growth Curve

-Unice­llular organisms divide by binary fission
-Each cell grows to full size, replicates its genetic material then divides into two identical daughter cells.
-By identical means, two cells divide into four, four into eight and so on, leading to an expone­ntial increase in cell numbers: 1 → 2 → 4 → 8 →2^n
-If we were to plot the number of cells in a population against time, we would get an expone­ntial curve
-Growth usually slows down due to:
a) supply of nutrients becoming exhausted
 
b) because metabolism leads to an accumu­lation of harmful waste substances
 
Lag Phase
-When an inoculum of bacteria is first introduced into some growth medium, it will probably require a period to adapt to its new surrou­ndings
 
-When an inoculum of bacteria is first introduced into some growth medium, it will probably require a period to adapt to its new surrou­ndings
 
-Eg, the carbon source in the medium is unfami­liar, the cells will need time to synthesise the necessary enzymes for its metabo­lism.
 
-Synth­esize molecules needed for protein synthesis and enzymes required for cell division
 
-no net increase in bacterial numbers, however the cells are metabo­lically active.
Length of the lag phase depend on:
a) age and general health of the cells in the inoculum
 
b) conditions of bacteria before transfer into growth medium
 
c) content of the growth medium
Log (expon­ential) Phase
-When the bacteria have acclim­atized to their new enviro­nment and synthe­sized the enzymes needed to utilize the available substr­ates, they are able to start regular division by binary fission.
 
-leads to the expone­ntial increase in numbers
 
-under optimal condit­ions, the population of cells will double in a constant and predic­table length of time, known as the ge­ner­ation (doubling) time.
 
-Cells are dividing at maximal rate
 
-Cells are most suscep­tible to the action of antibi­otics and other delete­rious agents
Stationary phase
-expon­ential phase is limited by enviro­nmental factors, and as the rate of growth slows down, the culture enters the next phase
 
-The levelling out of the growth curve does not mean that cell division has ceased comple­tely, but rather that the increase due to newly formed cells is cancelled out by a similar number of cell deaths.
 
-Occurs when the number of viable cells stops increasing
 
-Due to nutrients being used up and/or toxic products accumu­lating from cell’s metabolism
 
-as the death rate increases, the overall numbers fall and we enter the final phase of growth.
Death (or de­cline) phase
-As cells die off and the culture is unable to replace them, the total population of viable cells falls.
 
-Expon­ential decrease in the number of viable cells