Application of Cell Culture and Tissue Models for Assessing Drug Transport

Abstract

Drug transport in the body is a necessary step, from dosage form administration to the pharmacological target of the drug substance. Absorption (A), distribution (D), metabolism (M) and Excretion (E), i.e. ADME properties of drug substances, all include elements of drug transport. Cell culture and tissue-based models are often used to predict drug ADME properties, and to gain mechanistic insight into these. In the present chapter, the kinetics of drug transport and transport via drug transporters is described. The most common cell culture model for studying intestinal transport, i.e. the Caco-2 cell model is described in detail, and protocols for culturing and studying Caco-2 cells are included as an Appendix. Drug transport via carriers and transporters are important for drug substance ADME properties, and proton-coupled drug transport via the amino acid and peptide transporters PAT1 and PEPT1 in Caco-2 cells are discussed. Renal and hepatic models are also mentioned, as well as in vitro models of the blood brain barrier, which are discussed in more details. Even though in vitro models are easy to use and provide relatively reproducible results, areas of concerns and potential pitfalls are highlighted.

Appendix: Culture and Application of Caco-2 Cells

Caco-2 cells from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ no.: ACC 169. the cells are proliferated and then frozen and kept in storage as stocks in a cryotank.

These Caco-2 cells have been shown to be optimal for use in 17–20 passages after a new thawing. Characterization studies of membrane transporters show that the cells can be used immediately after thawing.

After a new thawing, it takes about a week of culture before the cells are ready for the first trypsinization when two cell vials of cryopreserved cells are cultured in a T75 flask.

1.1 Microscopy

All flasks are controlled by light microscopy prior to every trypsinization or change of medium. This is to monitor the growth of the cells and to adjust condition.

1.2 Preparation of Growth Medium

1.2.1 Composition

DMEM ⊕:
Penicillin/streptomycin 10,000 U/mL/10 mg/mL (Pen/Strep)	5.0 mL
l-Glutamine (dissolve precipitate by gentle shaking) (l-Glu)	5.0 mL
Non-Essential Amino Acids (NEAA)	5.0 mL
DMEM ad.	500 mL
DMEM ⊕ + 10 % FBS:
Foetal bovine serum (FBS)	50 mL
DMEM⊕ ad.	450 mL

1.2.2 Aseptic Preparation of the Medium

Thaw FBS, pen/strep and l-Glu in a water bath at 37 °C and shake gently before use. Heat NEAA in a water bath to 37 °C.

Add 5 mL Pen/Strep, 5 mL NEAA and 5 mL l-Glu to a bottle of 500 mL DMEM.

This solution is referred to as DMEM ⊕.

Transfer 50 mL FBS to a sterile 500 mL bottle or to the bottle containing excess DMEM ⊕ from the previous medium preparation, then fill up with DMEM ⊕ to a final volume off 500 mL (termed DMEM ⊕ + 10 % FBS)

1.3 Trypsinization Procedure (Sub-cultivation)

This is normally carried out at a confluence of approx. 90 %.

Instructions for trypsinization of a 75/175 cm² culture flask (T75/T175 flasks):

The cell culture should be handled aseptically in a LAF-bench.

• 1. Pre-warm the DMEM ⊕ + 10 % FBS, PBS and Trypsin-EDTA in the 37 °C water bath (approx. 15 min). Trypsin-EDTA × 3 may be used.

• 2. Control the culture flask visually and subject it to microscopy in order to check the cell layer for normal growth.

• 3. Remove the culture medium with a Pasteur pipette without touching the cell layer. Rinse the culture with 10/30 mL 37 °C pre-warmed PBS by gently moving the flask back and forth. Then remove the PBS with a Pasteur pipette.

• 4. Shake the trypsin-EDTA solution gently before use. Measure out 1 mL/T75 and 3 mL/T175 (0.5 mL/T25) and add this to the culture flasks by pouring it down the sides of the bottle. After this is done, move the flask so as to distribute the trypsin over the entire culture.

• 5. Leave the culture flask in the incubator for approx. 10 min.

• 6. By holding the flask perpendicular you will be able to see whether the cells have loosened, i.e. whether the trypsinization has lasted long enough. You can also check this by microscopy.

• 7. Trypsinization is stopped by pouring 10/20–30 mL DMEM ⊕ + 10 % FBS over the loosened cells on the bottom of the flask (Serum inactivates the trypsin-EDTA).

• 8. Aspirate the medium approx. 4–6 times with the pipette to separate any cell clumps (they can be a little difficult to separate in single-cell suspension. Further aspiration may be necessary for separation of cell clusters).

• 9. The cell concentration is determined by transferring a small aliqot (9 μL) of the cell suspension to a MultiCount 10 disposable counting slide using a Pasteur pipette. It may be necessary to dilute the cell suspension further, if the cell concentration is too great (an optimum count figure is approx. 100 cells/9 fields). Perform the counting using a conventional light microscope. Count at least 2 × 9 fields (3 × 3 fields—magnification X 10, see SOP No. 269-B). The different counts must be approximately equal.

• 10. The cell density in the suspension is calculated as follows:

  Average number of cells/9 fields * 10,000 = number of cells/mL.

• 11. A new passage is established by diluting the suspension to the desired concentration.

1.4 Seeding

Make sure the cells are evenly suspended in the pipette

1.4.1 Seeding in T-75 and T175 Flasks

The number of cells needed for seeding is dependent on passage No. Cell division is slower at the lower passage numbers. Therefore it is a good idea to seed a few more cells in the flasks when carrying out trypsinization during the first weeks after thawing.

Normally, the numbers of cells shown below are seeded, but with older passages it makes good sense to reduce the number of cells/flask a little:

Seed:
T-75	2.6 × 105 cells/T75	+13 mL DMEM ⊕ +10 % FBS
T-175	6 × 106 cells/T175	+33 mL DMEM ⊕ + 10 % FBS

Change medium every second day and trypsinize the flask again 1 week later.

Calculation:

$$ Number\ of\ mL\ \left( cell\ susp.\right)=\frac{2.6\times {10}^5\ \left(\frac{cells}{flask}\right)}{conc.\ of\ cell\ susp.\ \left(\frac{cells}{mL}\right)} $$

$$ Number\ of\ mL\ \left( cell\ susp.\right)=\frac{6\times {10}^5\ \left(\frac{cells}{flask}\right)}{conc.\ of\ cell\ susp.\ \left(\frac{cells}{mL}\right)} $$

1.4.2 Seeding on Filters

Most often T12-transwells of polycarbonate from Costar are used (T12-cci3401). These filters have a pore size of 0.4 μm, a growth area of 1.12 cm² and a diameter of 12 mm.

Various types of filters exist (see www.corning.com/lifesciences).

1.4.3 12-Well Filters (T12)

Prepare a cell suspension of 2.0 × 10⁵ cells/mL. Of this, 0.5 mL is added apically, and 1.0 mL DMEM ⊕ + 10 % FBS is added basolaterally.

(Use 12 × 0.5 = 6 mL cell suspension. Prepare a total of 7 mL with 1.4 × 10⁶ cells for 12 wells, corresponding to 1.0 × 10⁵ cells /filter or 8.93 × 10⁴ cells/cm²).

Calculation:

$$ Number\ of\ mL\ \left( cell\ susp.\right)=\frac{2\times {10}^5\ \left(\frac{cells}{mL}\right)\cdot total\ number\ of\ mL}{conc.\ of\ cell\ susp.\ \left(\frac{cells}{mL}\right)} $$

Cells seeded on filters are normally used for experiments on days 11–25, the cells must always be used for experiments the day after they have had their medium changed.

1.4.4 Seeding in Trays

1.4.5 12-Well Tray (B12)

Prepare a cell suspension of 2.26 × 10⁵ cells/mL. Add 1.5 mL of this per well.

(Use 12 × 1.5 = 18 mL of cell suspension. Prepare a total of 20 mL with 4.52 × 10⁶ cells for 12 wells, corresponding to 3.39 × 10⁵ cells/well or 8.93 × 10⁴ cells/cm²).

Calculation:

$$ Number\ of\ mL\ \left( cell\ susp.\right)=\frac{2.26\times {10}^5\ \left(\frac{cells}{mL}\right)\cdot total\ number\ of\ mL}{conc.\ of\ cell\ susp.\ \left(\frac{cells}{mL}\right)} $$

Cells must always be used for experiments the day after they have had their medium changed.

1.5 Change of Medium

1.5.1 T75/T175 Flasks

Remove the medium with a Pasteur pipette connected to a tube with vacuum suction.

After this, replace the medium with 13/33 mL DMEM ⊕ + 10 % FBS

1.5.2 6 and 12-Well Filters

• Remove the medium with a Pasteur pipette connected to a tube with vacuum suction.

• Empty the wells of medium, first basolaterally and then apically. This must be done without touching the cell layer on the filter (Avoid leaving the cells without medium for too long).

• Then replace the medium with fresh DMEM ⊕ + 10 % FBS, first apically and then basolaterally with:

  6-well filters:	apically:	2.0 mL
  	basolaterally:	2.5 mL
  12-well filters:	apically:	0.5 mL
  	basolaterally:	1.0 mL

1.5.3 6, 12, 24 and 96-Well Trays

Cells are seeded in the wells once a week. After this, the medium must be changed every other day until the cells are to be used.

• Remove the medium with a Pasteur pipette connected to a tube with vacuum suction. This must be done without touching the cell layer on the bottom.

  (Avoid leaving the cells without medium for too long)

• After this, replace the medium with fresh DMEM ⊕ + 10 % FBS with:

  6-well:	3.0 mL
  12-well:	1.5 mL
  24-well:	1.0 mL
  96-well:	0.2 mL

1.6 Freezing Procedure

The freezing of cells is done in an ordinary freshly prepared growth medium DMEM ⊕ with 15 % FBS, 5 % DMSO added, where the cell concentration is 2 × 10⁶ cells/mL, corresponding to a cryotube.

In order to protect the cells during freezing, all work done in the period during which the cells are affected by DMSO must be carried out as quickly as possible. The same applies during the thawing procedure.

Preparation of medium for freezing:

1.	90 % DMEM ⊕ + 10 % DMSO	(9 mL DMEM ⊕ + 1 mL DMSO)
2.	70 % DMEM ⊕ + 30 % FBS	(7 mL DMEM ⊕ + 3 mL FBS)

NOTE: DMEM ⊕ + DMSO must be prepared aseptically and sterile-filtered before use.

1.6.1 Final Concentration in Freezing Medium

DMEM ⊕	80 %
DMSO	5 %
FBS	15 %
Cells	2 × 106 cells/mL

1.7 Work Procedure

Normally four (or more) extra T175 flasks are seeded the week before in connection with the trypsinization.

• 1. The trypsinization procedure is as usual.

  The total number of cells is calculated.

• 2. Then calculate how many cryotubes can be frozen (2 × 10⁶ cells/tube).

• 3. Put the cells in 15 mL centrifugal tubes with conical bottoms (max. 10 mL cell suspension/tube). Each tube can contain, for instance, 8 × 10⁶ cells, corresponding to four cryotubes.

  (There has to be at least 1 mL medium in which the cells can be resuspended after they have been centrifuged. This means that there must be enough cells for at least two cryotubes/centrifugal tubes)

  Calculation:

  $$ Number\ of\ mL\ \left( cell\ susp.\right)=\frac{8\times {10}^6\ \left(\frac{cells}{mL}\right)}{conc.\ of\ cell\ susp.\ \left(\frac{cells}{mL}\right)} $$

  The tubes must all contain the same amount of medium. If there are an odd number of tubes, prepare an extra tube containing an equal amount of water. Place the tubes in pairs opposite one another in the centrifuge.

• 4. Centrifuge the cells at approx. 1000 g for 10 min −4 °C.

• 5.

  When preparing cryotubes, mark them with:	Cell type
  	Date
  	Passage No.
  	Cell concentration
  	Your initials

  (Increase the passage No. by 1 in connection with the trypsinization, so that the new passage no. is noted on the cryotube. On thawing, note the new passage no. on the T75 flask.)

• 6. Carefully remove the supernatant from the centrifugal tube using suction.

• 7. Carefully resuspend the cells in 1 mL, corresponding to two cryotubes, DMEM ⊕ + 30 % FBS per tube (mL DMEM ⊕ + 30 % FBS depends on how many cells are in the centrifugal tube)

• 8. Transfer the cell suspension to two cryotubes (with a silicon gasket), each containing 0.5 mL.

• 9. Carefully add 0.5 mL DMEM ⊕ + 10 % DMSO drop wise to each cryotube.

  The cryotubes are placed in a special freezing box, Nalgene® Cryo 1 °C Freezing Container (which is usually located in the refrigerator outside the cell room). The freezing box can hold a maximum of 18 cryotubes per freezing. Alternatively, a specially-made polystyrene box can be used for the freezing.

  The freezing box/polystyrene box is so adjusted that freezing takes place at a rate of 1 degree/min.

  Place the freezing box/polystyrene box in the −80 °C freezer for a minimum of 2 h.

• 10. After freezing at −80 °C for at least 2 h, the cryotubes are moved to one of the cryotanks.

  This is done either by placing 4–5 cryotubes in a cane (a holder specially made for cryotubes), which is then placed in the appropriate section of the cryotank (small/medium)

  The cryotank is divided into several sections, which can each contain a certain number of canes. Number all canes in a section consecutively, and write these numbers with a marker pen on the broad end. Each cane can hold a maximum of six cryotubes.

  Each cell line has a special section in the cryotank, which can be seen in the freezing folder.

  Or transfer the cryotubes to a special freezing box (which holds 96 tubes) and place in one of the holders in the large cryotank.

• 11. The freezing tables must be filled in with all the relevant information in the folder for the cell stock.

All work with cells from item 7 to 10 must be carried out as quickly as possible to avoid the cells being damaged during freezing and the transferal to the cryotank .

1.8 Thawing Procedure

When thawing new cells received from the DSMZ, follow the thawing procedure found in the “product information sheet” that comes with them.

With other thawing, follow the thawing procedure below.

1.8.1 Work Procedure

• 1. Add 13 mL of DMEM ⊕ + 10 % FBS to a T75 flask and place it in the incubator for approx. 15 min (a T25 with 5 mL DMEM ⊕ + 10 % FBS can also be used).

• 2. Remove the cells from the cryotank and thaw them in a small beaker containing autoclaved water warmed to 37 °C in the water bath (It is important that the thawing is done as quickly as possible, and for this reason the beaker containing 37 °C water must be brought to the cryo tank).

  A face shield must be worn, as the cryo tube can burst during thawing.

• 3. Immediately after thawing, carefully transfer the cells to a culture flask, T75 (T25).

  Write the data from the cryo tube on the flask.

• 4. The cells must have their medium changed the day after thawing.

• 5. After this, the medium is changed every other day (Mon.-Wed.-Fri.), until the cell layer is confluent, after which they are trypsinized.

• 6. Write in the folder for the cell stock when the cells were thawed and how the cell growth proceeded up until the date of trypsinization.

1.8.2 Passage Nomenclature

Passage = sub-cultivation.

The passage numbers consist of:

Number1-(number2)-number3

Number1

• Passage Number as the cell line is received—if this number is 1—the cell line is received without a passage number.

( ) + Number2

• In ( ) is info on freezing. Number2 is the number of trypsinizations before freezing—if there are more numbers in the brackets means that the cell line is proliferated several times.

Number3

• Number of trypsinizations since thawing.

Example:

Received without passage No.—proliferated by two trypsinizations before freezing:
New passage No. at thawing:	1—(2)—0
After first trypsinization:	1—(2)—1