细胞培养技术

Passaging cells

Pour out media from flasks.

Wash with Hanks. 5 ml per flask.

Tilt around then dump.

Add 4 ml of Trypsin / EDTA to each flask. Tip, then bang.

Add 20% FBS NCTC media and tilt. 4 ml per flask.

Scrape (most of the small cells are already released; proliferative cells lift up easy, differentiative cells are more adherent).

Place contents into a 50 ml Falcon tube.

Spin at 1000 RPM for 5 min (#5 setting = 1000)

Dump off supernatant.

Add enough media to allow 2 ml of cells per flask (RCHO-cells start to differentiate at day two. At that time a flask contains ca. 1 million cells. They are spread by 1 : 3).

Each flask should have 8 (or 10) ml of 20% FBS NCTC media + 2 ml of cells for a total of 10 (or 12) ml.

Freezer and Cell Line Database

Very useful Microsoft Access database program (486kb) for managing your cell lines stored in freezer. It's easy to use and has a very nice screen.

20040724154009292.rar

Defrosting Eukaryotic Cells Frozen In Liquid N2

Reagents / Solutions

• Frozen cell line.
• Growth media (e.g. RPMI1640)
• Foetal calf serum (FCS).
•  

Protocol

1. Make 20ml growth media/10% FCS for each vial of cells to be thawed.

2. Place a small sandwich box containing warm (~ 37℃) water in the CO₂ incubator.

3. Remove vial(s) of cells and thaw rapidly by placing in an expanded polystyrene 'floater' in the water bath. Transfer cells to a universal.

4. Add 10ml media/10% FCS very slowly. First ml should take ~ 1 min, second ml should take ~ 45 seconds etc.

5. When cells are resuspended in 10ml spin down in bench-top centrifuge and resuspend in 10 ml media/10% FCS (this does not need to be added slowly).

6. Place cells in large flask and allow to settle for ≥ 2 hours.

7. Take a sample, assess % viability and cell number.

8. Allow to grow overnight.

9. Assess cell number and viability, if the cell density is too high for the normal grwoth of the cell line add further normal growth media (e.g. RPMI1640/10% FCS.

Cell Freezing

Reagents / Solutions

• Cells (at least 5 x 10⁶).
• Foetal calf serum (FCS), heat inactivated at 65℃ for 1 hr.
• Dimethylsulfoxide (DMSO).
• Freezing vials.
• Hi - Tech Cell freezer (polystyrene box wadded with cotton wool ).
• Liquid nitrogen bank. 

Protocol

Spin down cells, remove and discard the supernatant.

Resuspend the cells at 5 x 10⁶ per ml in FCS.

Add DMSO to 10% (by volume) dropwise, agitating gently after each drop. Do this slowly.

Place 1ml aliquots into the vials. Remember to label the vials precisely.

Put vials into central positions of a polystyrene rack (from e.g. Boehringer Enzyme Storage box), and place that rack into an expanded polystyrene box (e.g. 'Eprack' as supplied by Scotlab) which is wadded with cotton wool.

Place lid on box and seal in place with tape.

Place box in the vapour phase of the liquid nitrogen bank. It must not be placed in the liquid phase. Leave for more than 4 hrs to freeze.

Remove vials from box and place in liquid phase in the cell bank.

Test the efficiency of the freezing procedure by thawing (method) and growing on one vial. 

If the cells were healthy (> 95% viable) at the start of this procedure, then we obtain a culture 80-90% viable 24 hours after thawing and growing on (method) the test vial from point 9.

Master Cell Bank

Author: Nanci Donacki Source: Contributed by Nanci Donacki Abstract: Provides detailed protocol for establishing a master cell bank

Purpose

To describe the preparation of a Master Cell Bank

Safety

See SP 09-001 for lab safety considerations for the cell culture lab.

Equipment

• Laminar Flow Hood
• Freezers, -70 ℃or Rate-Controlled Freezer
• Liquid Nitrogen Freezer
•  

Materials

• Cryovials, 1.8 ml (Nunc or equivalent)
• Cryovial rack (Nunc or equivalent)
• Sterile Centrifuge tubes, 50 ml (VWR # 21008-146 or equivalent)
• Fetal Bovine Serum, heat inactivated (BioWhittaker # 14-503F or equivalent)
• Sterile DMSO (Sigma # D2650 or equivalent)
• Sterile Pipets of appropriate sizes
• Ice
• Permanent Marking Pen
• Lab Coat
• Latex Gloves
• 70 % alcohol or equivalent
• Trypan Blue, 0.4% (GIBCO # 630-5250AG or equivalent)
• Hemocytometer
• Nunc Freezing Container (# 5100-0001)
• Freezer Log
•  

Procedure

• The Master Cell Bank consists of 20 vials of cells at 5 x 10⁶ cells/vial.
• Before preparing the Master Cell Bank, ensure that the cell line is stabilized and that the cells are free from mycoplasma contamination.
• The day before freezing, refeed the cells with fresh medium, or add additional medium to suspension cultures to ensure that they are in log phase of growth.
• Chill the Freezing medium on ice.
• Label with cryotubes with the cell line name, the cells/vial, and the date.cells/ml. Resuspend the cells in the freezing medium by gently pipetting.
• Place the tube on ice. Dispense the cells, 1 ml/vial, into the labeled cryovials. Recap vials tightly. Complete all vials.
• Place the vials into the Nunc Freezing Container or Rate Controlled Freezer Racks.
• Replace the lid on the freezing container. Place the Freezing Container in the -70℃ Freezer overnight.
• Transfer the vials to the vapor phase of the liquid nitrogen freezer.
• Record the cell line information and location in the Freezer Log.

Freezing Cells

Contributor: Suprya Jayadev Date: January 10, 1991

1) Keep prepared solutions on ice.

2) Determine total cell count of cells to be frozen. (e.g. 1 X 10⁸ )

3) Determine number of vials to be frozen. (e.g. 10 vials at 1 X 10⁷ )

4) Centrifuge cell suspension.

5) Resuspend cell pellet in half the required volume of freezing media. (eg if freezing 10 vials, add 5 mls freezing media)

6) Keep cell suspension on ice.

7) Slowly add dropwise with mixing an equal volume of DMSO solution.

8) Dispense 1 ml of final mixture per sterile Nunc freezing vial.

9) Freeze in liquid nitrogen plug or in controlled rate Planar freezer.

Freezing media

60 ml RPMI1640 or DMEM

40 ml Gammaglobulin free Horse Serum (or FCS)

1 ml Penicillin - Streptomycin

Filter through 0.2µ filter.

Aliquot in 15 ml conical tubes and store in -200 C freezer for long term periods.

Aliquots can be stored in tissue culture flasks in refrigerator for short periods.

20% DMSO solution MAKE FRESH ON DAY OF USE!

If using 100% DMSO:

4 ml pH adjusted medium RPMI (orange-red color)

1 ml sterile 100% DMSO

0.1 ml versene

Filter through a 0.2 µ filter.

If using 50% DMSO:

3 ml pH adjusted medium

2 ml 50% DMSO

0.1 ml versene

Filter through a 0.2 µ filter.

NOTE: Versene can be left out, but adding it prevents cells from clumping.

Freezing and Thawing of Mammalian Cell Lines

For long term storage of myeloma cells, hybridoma cells, T cells, and other mammalian cell lines in liquid nitrogen, and restoring them in culture.

Freezing

Preparation

Cells are to be frozen in liquid nitrogen, so make sure your canisters are relatively full of nitrogen and you have room. Cells should be healthy (>90% viability) and growing in log phase. You will also require sterile 1 mL cryo-vials; they have a screw-top and rubber seal to keep the nitrogen out.

Freezing

Count cells in a hemocytometer. Centrifuge 10 mL of cells on "2" in a clinical centrifuge for 10 minutes and resuspend in freezing medium (10% DMSO, 20% FCS, 70% media that you used to grow the cells such as RPMI or DMEM) at a concentration of 2 X 10e6 cells/0.5 mL freezing medium. Aliquot in cryo-vials 0.5 mL/vial.

Place vials upright in a styrofoam box and cover well. Place in a -70℃ for 24 hours.

Place vials in a wand and put in a liquid nitrogen container. 

Thawing

Preparation

Warm water bath to 37 ℃.

Place 10 mL media (RPMI, DMEM) in a sterile 15 mL centrifuge tube. Layer 2 mL FBS to the bottom of the tube, slowly, so that you can see two layers.

Make your growth media for your new culture. For monoclonals this would be DMEM with 20% FBS and penicillin-streptomycin.

Thawing

Take your cells out of nitrogen storage and thaw rapidly by swirling in the 37 ℃ water bath.

Sterilize the outside of the vial with 70% ethanol, bring in to the culture hood and add slowly to the top of the layered media you prepared. The cells should fall to the interface between the media and the FBS.

Centrifuge on "3" for 10 min. in a clinical centrifuge.

Pipet of the supernatant and resuspend in the growth media of choice you prepared earlier. Pipet into a T25 and place in a CO2 incubator for growth of your new culture.