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USE OF DIFFERENT CELL LINE FOR

ABSORPTION STUDY

VISHAL GANDAQ.A.T 1ST

PARUL INSTITUTE OF PHARMACY

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o cell culture is the complex process by which cells are grown under controlled conditions, generally outside of their natural environment.

o By this process prokaryotic,eukaryotic or plant cells are grown under controlled condition but in practice it refers to the culturing of cells derived from animal cells.

INTRODUCTION

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o Cell culture was first successfully undertaken by Ross harrison in 1907

o In 1885 Roux first time maintained embryonic chick cells in cell culture.

o 1911: Lewis and Lewis made the first liquid media consisted of sea water, serum, embryo extract, salts and peptones. They observed limited monolayer growth

o 1913: Carrel introduced strict aseptic techniques so that cells could be cultured for long periods.

o 1952: Gey established a continuous cell line from a human cervical carcinoma known as HeLa (Helen Lane) cells.

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o 1927: Carrel and Rivera produced the first viral vaccine - Vaccinia.

o 1933: Gey developed the roller tube technique.

o 1982: Human insulin became the first recombinant protein to be licensed as a therapeutic agent.

o 1985: Human growth hormone produced from recombinant bacteria was accepted for therapeutic use.

o 1987: Tissue-type plasminogen activator (tPA) from recombinant animal cells became commercially available

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o First development was use of antibiotics which inhibits the growth of contaminants.

o Second was the use of trypsin to remove adherent cells to subculture further from the culture vessel.

o Third was the use of chemically defined culture media.

MAJOR DEVELOPMENT IN TECHNOLOGY

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o MODEL SYSTEM: for studying basic cell biology, interaction between disease causing agent and cells, effect of drugs on cells, process and triggering of aging & nutritional studies.

o TOXICITY TESTING: in study of effect of new drugs.

o CANCER RESEARCH: study function of various chemical, virus & radiation to convert normal culture cell to cancerous cells.

cell culture used in different areas

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o VIROLOGY: cultivation of virus for vaccine production.

o GENETIC ENGGINERING: production of commercial protein, large scale production of viruses for use in vaccine production.

o GENE THERAPY: cells having a functional gene Can be replaced to cell which are having nonfunctional gene.

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o 3 Types of cell cultureso 1)Primary cell culture: This is the maintenance of

growth of cells dissociated from the parental tissue (such as kidney or liver) using mechanical or enzymatic methods, in culture medium using suitable glass or plastic containers. The primary cell culture could be of two types depending upon the kind of cells in culture.

Adherent cells - Cells shown to require attachment for growth are said to be anchorage dependent cells. The adherent cells are usually derived from tissues of organs such as kidney where they are immobile and embedded in connective tissue.

TYPES OF CELL CULTURE

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Suspension cells - Cells which do not require attachment for growth or do not attach to the surface of the culture vessels are anchorage independent cells/suspension cells. All suspension cultures are derived from cells of the blood system because these cells are also suspended in plasma in vitro e.g. lymphocytes.

o 2)Secondary cell cultures: When a primary culture is sub-cultured, it becomes known as secondary culture or cell line. Subculture (or passage) refers to the transfer of cells from one culture vessel to another culture vessel. This is periodically required to provide fresh nutrients and growing space for continuously growing cell lines. The process involves removing the growth media and disassociating the adhered cells (usually enzymatically). Such cultures may be called secondary cultures.

o  3)Cell Line: A cell line or cell strain may be finite or continuous depending upon whether it has limited culture life span or it is immortal in culture. On the basis of the life span of culture, the cell lines are categorized into two types:

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Finite cell lines - The cell lines which have a limited life span and go through a limited number of cell generations (usually 20-80 population doublings) are known as finite cell lines. These cell lines exhibit the property of contact inhibition, density limitation and anchorage dependence. The growth rate is slow and doubling time is around 24-96 hours.

Continuous cell lines - Cell lines transformed under laboratory conditions or in vitro culture conditions give rise to continuous cell lines. These cell lines show the property of ploidy (aneupliody or heteroploidy), absence of contact inhibition and anchorage dependence. They grow either in a monolayer or in suspension (see below). The growth rate is rapid and doubling time can be 12-24 hours.

Monolayer cultures - When the bottom of the culture vessel is covered with a continuous layer of cells, usually one cell in thickness, they are referred to as monolayer cultures.

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Suspension cultures - Majority of continuous cell lines grow as monolayers. Some of the cells which are non-adhesive e.g. cells of leukemia or certain cells which can be mechanically kept in suspension, can be propagated in suspension.

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o HUMAN CELL LINES:• DU145 (prostate cancer)• THP-1 (acute myeloid leukemia)• MCF-7 (breast cancer)• Saos-2 cells (bone cancer)• PC3 (prostate cancer)o PLANT CELL LINES:• Tobacco BY-2 cell o Rat tumor cell lines:• GH3 (pituitary tumor)• PC12 (pheochromocytoma)o Mouse cell lines:• MC3T3 (embryonic calvarium)

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o Choice of media depends on the type of cell being cultured

o Commonly used Medium are GMEM, EMEM,DMEM etc.

o Media is supplemented with antibiotics viz. penicillin, streptomycin etc.

o Prepared media is filtered and incubated at 4 C.

CULTURE MEDIA

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o Laminar cabinet-Vertical are preferableo Incubation facilities- Temperature of 25-30 C

for insect & 37 C for mammalian cells, co2 2-5% & 95% air at 99% relative humidity. To prevent cell death incubators set to cut out at approx. 38.5 C

o Refrigerators- Liquid media kept at 4 C, enzymes (e.g. trypsin) & media components (e.g. glutamine & serum) at -20 C

o Microscope- An inverted microscope with 10x to 100x magnification

o Tissue culture ware- Culture plastic ware treated by polystyrene

BASIC EQUIPMENT USED IN CELL CULTURE

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o Possibly keep cultures free of antibiotics in order to be able to recognize the contamination

o Never use the same media bottle for different cell lines. If caps are dropped or bottles touched unconditionally touched, replace them with new ones

o Necks of glass bottles prefer heat at least for 60 secs at a temperature of 200 C

o Switch on the laminar flow cabinet 20 mts prior to start working

o Cell cultures which are frequently used should be subcultered & stored as duplicate strains.

SAFETY ASPECTS IN CELL CULTURE

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o Use actively growing cells that are in their log phase of growth, which are 80-90% viable

o Keep exposure to trypsin at a minimumo Handle the cells gently. Do not centrifuge

cells at high speed or roughly re-suspend the cells

o Feeding & sub culturing the cells at more frequent intervals then used with serum containing conditions may be necessary

o A lower concentration of 104cells/ml to initiate subculture of rapidly growing cells & a higher concentration of 105cells/mlfor slowing growing cells

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o Several cultured epithelial cell lines of human or animal origin can be used as in vitro drug absorption models to predict the absorption of compounds across intestinal tissue in vivo.

o The Caco-2 human colonic cell line is perhaps the most widely used cell line for this purpose. .

o Caco-2 cells adopt a polarized morphology and express many intestinal transport proteins and other enzymes characteristic of epithelial cells of the small intestine.

o Absorption Systems offers:o 1)In vitro assays for absorption across human or animal

intestinal strips o 2)Oral bioavailability studies in rodents, dogs, pigs and

non-human primates o 3)Intestinal loop perfusion studies in rats

ABSORPTION STUDY

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o The Caco-2 cell line is an immortalized line of heterogeneous human epithelial colorectal adenocarcinoma cells.

o Caco-2 cell monolayers spontaneously differentiate to express morphological and functional characteristics of mature small-intestinal enterocytes. The differentiated monolayers are polarized, with microvilli on the apical side.

o it is developed by the Sloan-Kettering Institute for Cancer Research

Caco-2 cell culture

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o Spontaneously differentiate to express morphological (polarized columnar cells) and functional characteristics of mature small-intestinal enterocytes.

o Four times higher in transepithelial resistance compared to HT 29-cell monolayer.

o It expresses various drug metabolizing enzymes like aminopeptidase, esterase, and sulfatase.

Advantage of Caco-2 monolayer

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o Tissue in the villus contains more than one cell type

o Dose not produce the mucus and unstirred water Observed in the intestine

o No P-450 drug metabolizing enzyme activity has been reported

o Expensive methodo Time consuming as 21 days required for full

cell differentiationo The necessity of LC / MS or HPLC for

quantitationo Influence of P-gp is difficult to estimate.

Limitation of CaCO-2 monolayer

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CaCO2 Culture media

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TRANSPORT MEDIA

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CELLULAR ARCHITECTURE

3 DAYS 21 DAYS

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o A. Trans Epithelial Electrical Resistance (TEER) measurements:

o Measurement of the integrity of the caco-2 cell monolayer

o After washing the cell monolayer with 37°C tempered D-PBS (with Ca2+, Mg2+) (Dulbecco's Phosphate Buffered Saline), 1600 μl transport medium was added into the apical and 2800 μl transport medium was added into the basal compartment.

o Allow for equilibrium for 60 min in the cell culture incubator.

BARRIER FUNCTION

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o 4. The measurement chamber was tempered to 37°C with transport medium before the measurement. For the post-experimental TEER measurement, the withdrawn volume in the apical compartment was replaced with transport medium before TEER was measured.

o o 5.Caco-2 monolayer with TEER values exceeding

250 Ωcm2 were used for transport experiments.o o 6. Background TEER may be recorded in wells

without cell monolayers, and can be subtracted from the raw TEER values with cells.

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o Rinse the monolayer three times with 300 µL HBSS ( Hank’s buffer saline solution) in the apical wells and 28 mL in the feeder tray.

Add 300 µL of LY solution to each well in the filter plate (Apical Template).

Add 600 µL HBSS to each well of a 24-well receiver tray (Basolateral Template).

Assemble the filter plate and 24-well receiver plate and incubate for 1–2 hours at 37°C.

Remove the filter plate from the receiver plate and place the receiver plate into a fluorescent plate reader. Determine the LY fluorescence using an excitation wavelength of 425-430 nm and an emission wavelength of 515-520 nm, 540 nm.

B. Lucifer yellow (LY) rejection:

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Calculate the percent of LY rejection across the cell monolayer by measuring fluorescence in the receiver plate as compared to an ‘equilibrium’ standard.

The standard plate should consist of 4 wells with 600 µl HBSS (blank) and 4 wells with 200 µl LY (100 µg/ml) + 400 µl HBSS (equilibrium samples)

calculate the LY rejection using the following equation:

LY rejection=100%-%ly passage

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Caco-2 human colon adenocarcinoma cells are grown to confluence and differentiated for three weeks on filters. Test agent is added to one side of the monolayer, and permeability is assessed by analyzing the concentration of the test agent on the other side of the monolayer using LC-MS/MS.

The following Caco-2 assays are available: Monodirectional: High permeability predicts good human oral

bioavailablity. Bidirectional: High efflux ratio indicates possible P-gp or other

transporter efflux. Bi-directional (P-gp substrate determination): Test agent is incubated on

either side of the monolayer in the presence and absence of a known P-gp substrate.

Bi-directional (P-gp inhibitor determination): Test agent is incubated on either side of the monolayer in the presence and absence of a known P-gp inhibitor.

Bi-directional (Other transporters): it require regarding the testing of your test agents to be substrates or inhibitors of other efflux transporters (e.g. BCRP, MRP1, MRP2, etc.).

PRINCIPLE

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Procedure: After the desired cell growth period, remove the plate from the

incubator and determine the electrical resistance for each well (as described above). Wash the monolayer, exchanging the volume three times using sterile HBSS, pH 7.4. After washing, remove the buffer from the filter plate and feeder tray.

Transfer the filter plate to a 24-well transport analysis plate. To determine the rate of drug transport in the apical to

basolateral direction, add 300 µL of the test compounds to the filter well. Drug concentrations typically ranging from 10 µm to 200 µm may be used (achieve desired concentration using HBSS, pH 7.4 or an alternative buffer of desired pH).

Fill the wells of the 24-well receiver plate with 600 µL buffer. To determine the rate of drug transport in the basolateral to

apical direction, add 600 µL of the test compounds to the 24-well receiver plate.

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Fill the filter wells (apical compartment) with 300 µL of buffer.

Join the filter and receiver plates once all drugs and buffer have been added. Begin timing the experiment.

Incubate at 37°C shaking at 60 rpm on a rotary shaker. Typical incubation times are 1 to 2 hours.

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CALCULATION OF APPEARENT PERMEABILITY

• Papp=Area * time *

[Drug]acceptor

[Drug]initial,donor

Where, VA= volume in ml in the acceptor well area =the surface area of the membrane time= total transport time in seconds

VA

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1) In Drug Discovery: To test the absorption profiles of the new molecular entities in the lead optimization state.

2) In pre-clinical drug development: US FDA recognizes Caco-2 to measure permeability as part of

the bioequivalence waiver process.

3) To evaluate effect of pharmaceutical excipients.

4) To study transport mechanism from many compounds

5) In drug metabolism & toxicity effects.

6) Others like study of CFTR, regulation of protein expression; genetics study.

APPLICATION

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Other model co-related with caco2

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PAMPA & caco-2 should not be considered as competing permeability methods.

Good correlation between PAMPA & caco-2 data for a compound indicates a predominance of passive diffusion in its permeation.

Lack of correlation indicates absorptive (active, paracellular ,gradient effect for

acids) or ecretarys (efflux, gradient effect for bases)

permeation mechanism

PAMPA VERSUS Caco-2 MODEL

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one of the commonly used cell monolayer systems to assess the human intestine barrier.

MDCK cell lines can reach full differentiation in 3-7days and are therefore relatively easy for cell culturing and assay maintenance.

DISADVANTAGE MDCK cell lines originate from dog kidney. The expression of transporters is quite different from human

intestine.

The Madin-Darby canine kidney (MDCK) cell model

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www. nutriweb.org www.uv.es/ www.researchgate.net

REFERENCES

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