Cryopreserved Hepatocytes Purification Kit

Kit Components
I). Tube A = Supplemented ISOM's¹ Media + Percoll™ Solution (Percoll™ is a Registered Trade Mark of Amersham-Pharmacia Inc.)

Package Content.................25.7 ml of supplemented ISOM's Media + 11.8 ml of Percoll™ in each of 2 x 50 ml conical tubes

II). Tube B = Supplemented ISOM's Media

Package Content.................45 ml of supplemented ISOM's Media in each of 2 x 50 ml conical tubes

Safety Recommendations

• When using this product, follow food laboratory safety procedures:
• Do not eat, drink or smoke.
• Avoid contact with skin or eyes.
• Do not inhale aerosols.
• Do not pipette by mouth.
• Wear suitable protective clothing, gloves and eye protection.
• Steam sterilize product or treat product with 1 % solution of Sodium Hypochlorite prior to disposal.

Percoll™ Centrifugation Instruction Manual

The following instructions describe procedures for thawing cryopreserved hepatocytes and subsequent purification using Percoll™ centrifugation. The Percoll™ centrifugation step can significantly increase the percentage of viable cells.² The kit will allow for two separate purifications. Up to 3 -1.5 ml cryo-vials can be purified in a single centrifugation procedure. The kit will allow for two separate purifications. Up to 3 -1.5 ml cryo-vials can be purified in a single centrifugation procedure. **

**NOTE: For optimal viable cell recovery, thawing one cryo-vial per single centrifugation procedure is recommended.

Equipment not included in Kit

1. 37° waterbath
2. Low speed centrifuge
3. Biosafety hood
4. Trypan blue (Sigma, catalog # T8154)
5. Hemacytometer
6. Microscope
7. 1X PBS
8. KHB Buffer (Sigma, catalog # K3753)

Steps for Thawing Cells and Percoll™ Centrifugation
(one to three - 1.5 ml Cryo-Vials per Centrifugation)

I. Preparation of Percoll™/ISOM's Media Solution

1. Remove 1 tube each of Tube A and Tube B from kit container:

a. If thawing human, mouse or rat hepatocytes, keep tubes A and B on ice.
b. If thawing dog or monkey hepatocytes, warm tubes A and B to 37 °C.

2. Add appropriate amount of Tube B (ISOM's media) to Tube A depending upon the number of cryo-vials used. Refer to the following table for mixing instructions:

II. Thawing Frozen Hepatocytes

NOTE: Thawed hepatocytes are fragile. Handle cells gently to maintain viability.

1. Cryo-Vials should be stored in Liquid Nitrogen vapor until ready for use.
2. Remove the cryo vial from liquid nitrogen storage and immediately place it in a 37°C water bath for approximately 1.5 minutes, or until the cells are nearly thawed. Remove the tube immediately from the water bath and place on ice.

NOTE: Immediately proceed to next step, as the cryoprotectant can be toxic to cells and delays at this point may decrease viability.

3. Using a 2 ml serological pipette, add 1.5 ml of the thawed hepatocytes to Tube A. Rinse the empty hepatocyte tube with ISOM's media (Tube B solution). Refer to the following table to determine the amount of ISOM's media needed for the rinse step (the amount of media depends upon the number of cryo-vials used). The final volume in Tube A should be 49.5 ml or 24% Percoll™ to achieve the most efficient Percoll™ separation. Mix Tube A by gently inverting 2-3 times.

Temperature Requirements for Individual Species

III. Centrifugation and Cell Re-Suspension

1. Centrifuge Tube A at 100 g for 5 minutes at 4°C (37°C for dog hepatocytes), using "Low-Brake" setting on centrifuge.
2. Aspirate and discard the supernatant fluid without disturbing the cell pellets.
3. Add remainder of Tube B (ISOM's media) to Tube A, slowly, along the side of the tube without disturbing the cell pellet.
4. Centrifuge at 50 g for 5 min at 23°C (37°C for dog and monkey hepatocytes), using "Low-Brake." Aspirate and discard the supernatant fluid.
5. Re-suspend the cell pellet in approximately 2 ml of pre-warmed KHB buffer. Gently tap to re-suspend. (The exact amount of KHB buffer needed for cell re-suspension may vary depending of the application and the number of cryo-vials processed). NOTE: For rat and mouse heptatocytes it is recommended to use KHB buffer supplemented with 10 mM Fructose and 3 mM Glycine.
6. Determine cell viability and viable density as mentioned below. This is your final cell concentration. Maintain the cells at 37°C.
7. The recommended final cell concentration is 1 to 2 x10e6 cells per ml KHB buffer. The cells can be further diluted as needed. The cells should be used immediately after the purification steps.
8. For cell incubations longer than 2 hours, it is recommended that Hepato-STIM media be used in place of KHB buffer (Hepato-STIM hepatocyte culture media is a BD Biosciences Discovery Labware product; Catalog No. 355056).
   

Determination of Cell Viability

a) Make a 1:10 dilution of trypan blue solution with 1X PBS buffer.
b) Make two 1:5 dilutions of the above cell suspension with the diluted trypan blue solution. (i.e. 40 ul of trypan blue solution + 10 ul of cells into an eppendorf tube).
c) Wait for about one minute, then immediately pipette 10 ul per tube into separate chambers of a hemacytometer.
d) Observe the cells under the microscope (100X magnification).
e) Count the total number of live cells (clear cells) and the dead cells (blue cells) in quadrants; A, B, C, D, for each chamber (each quadrant has 16 small squares, each separated by triple lines, the middle one of which is the boundary). A B D C

f) Calculate the % viability and average cells/ml (c/ml*e6) concentration below.

% Viability = { live cells/ total cell (live+dead)} x 100

Viable Cells/ml x 10e6 = Viable Cell No./4 x (Cell Dilution Factor) x 10e4

References

1. Isom, et al., Proc. Natl. Acad. Sci. 81:6378-6382, 1984
   
2. Kreamer, et al., In Vitro Cell Dev. Biol. 4:201, 1986