Protocol-Cell Thawing/Cell Freezing Protocol - 国外信息动态和...

来自 : 发布时间：2025-07-10

Today I will share some protocols on cell thawing and freezing
本帖最后由 linxingxing 于 2009-10-17 20:04 编辑
Cell Thawing/Cell Freezing Protocol $ Y6 G\" m$ |1 ^1 t& M\' [
Freezing Cells:
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1 Cells should be growing well or known to be in log phase
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2 Count, collect and pellet cells in a 15mL test tube
3 Resuspend in freezing media so that the concentration is no more than 5x10^6 cells/mL of cold freezing media - p\" |\" @. E; e# m
4 Transfer 1mL of cells to appropriately labelled cryovials and maintain on ice for approximately 30minutes 7 G/ Q$ u# W\" q5 f& [) RK\' T
5 Transfer vials to -80C freezer for 24hrs ^) A\' {& M6 S1 L- J+ H
6 Transfer to liquid nitrogen dewar or -140C freezer for long-term storage. 8 }1 |\' T+ `- v5 v+ P
7 Freezing media
10% DMSO # D5 V\" m5 Y) xC! ~. A
90% FCS
you\'ll need 1mL per 5x10^6 cells
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Thawing Cells:
1 Remove vial from Liquid Nitrogen or -140C freezer and immediately transfer to 37C water bath \" X. f1 Y& |4 d
2 While holding the tip of the vial, gently agitate the vial, being careful hnot to allow water to penetrate the cap or seal ( u|$ a/ |4 V) b2 C& [
3 When completely thawed, transfer contents of vial to 15mL test tube \' o, `# g% M2 S4 w, _; g
4 Slowly add 10mL warm complete media and spin at 1000g for 5min * Y0 z3 t7 |3 T. C4 f
5 Decant media and resuspend pellet in a volume of complete media appropriate for flask or macrowell
6 Transfer cells to flask or 24 well plate and incubate at 37C and 5%CO2 * y9 x- F9 h) c\" M, ~, t+ U\" B* e
7 Cells can be checked visually or counted, beginning at approximately 1hr, for an estimate of viability. Immediate cell counts can be misleading
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from: The University of Chicago
3 Add 7ml Media
4 Pipette up and down to distribute cells throughout media (i.e. not clumped together) v9 Q2 Q/ M1 t: C3 A
5 Add media to sterile falcon tube (15ml = 1 flask, 50ml = 5 flasks) ; k8 o# t\' e! i( s7 |3 B
6 Spin down 1-2K, 5 mins ! Z6 Z t3 l/ R
7 Take off media \" H* M# M5 @\" F/ x, L# K, c
8 Resuspend pellet in 9ml FCS and 1ml DMSO
9 Distribute in 1ml aliquots (10 cryovials) ) u9 w~+ @8 k~+ n( Sq0 s\" B! z
10 Move cells to -84oC overnight wrapped in cotton wool in a polystyrene box
11 Finally freeze cells in liquid N2
Unthawing
1 Warm DMEM in waterbath 6 Q$ E! [& N\" m5 n; }
2 Thaw cryovial at 37oC quickly until cells become molten.
3 Aliquot the 1ml of cells using a disposable pipette into 10ml fresh media in a TC flask9 `& s2 p\" ab1 a! k$ `
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from: Standard Protocols
Freezing
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It is best to freeze cells that are growing rapidly. With adherent cells, it is easiest to set up 100 mm dishes, give them fresh medium the day before you freeze them, and freeze them just as they become confluent. 4 O) L) h% g4 ~/ Y3 t1 E
If you are working with suspension cells, make sure that they are growing vigorously before you freeze them. * v0 E5 B( b2 B. L; Y; {2 S/ i# T
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1. Trypsinize the cells if necessary, and then spin them down. 5 E\' u( ?1 {8 E& H7 F+ Lv0 K! X
Resuspend them in one half the volume that you want to end up with.8 l* B4 L- j! L
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In other words, use 0.5 ml per 100 mm dish you are using.5 l\" g; H/ U+ b3 K0 b
The cells should be resuspended in DMEM containing 20% fetal calf serum.
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Put the tube in an ice bucket and let the cells cool.
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2. Now, add the DMSO to the cells slowly. This step is accomplished by adding to the resuspended cells an equal volume of DMEM containing 20% fetal calf serum and 20% DMSO.
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This should be done dropwise and the cell suspension should be mixed by swirling after the addition of each drop.
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The reason to do this slowly is to avoid osmotic shock and to minimize the heating that occurs when DMSO is added to water. When you are finished, you should have the cells suspended in DMEM containing 20% fetal calf serum and 10% DMSO with all the cells from a single 100 mm dish in 1 ml.
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3. Chill the cells on ice, in the centrifuge tube, for exactly 30 min. Then transfer them to freezing vials which have been pre-chilled in the freezer in the plastic rack, which has also been pre-chilled in the freezer. Use 1 ml per vial. # q% O6 ]5 I8 v% S
4. Put the vials in a well insulated box that has also been pre-chilled in the freezer. Insulation is best accomplished with cotton. % j, T4 w. i2 W
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Then put the box in the -70°C freezer overnight, for slow cooling, and then put them into liquid nitrogen.
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Cells will store forever in liquid nitrogen. Storage at -70°C is riskier.$ }\' h2 B( ]! W+ |! k* Q
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Thawing.\' H5 X& o% g5 g
Cells should be thawed rapidly and then diluted slowly into warm growth medium. / v2 D0 k1 O\' ~9 G0 l! _
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The best way to do this is to transfer the contents of the thawed vial to a 50 ml centrifuge tube and then add--dropwise, with continual swirling--10 ml of warm medium.
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Then put the cells on a 100 ml dish. This way the cells don\'t suffer osmotic shock.) Nf9 i, U8 Q* c0 I& E) d
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Some cells seem to benefit from being diluted into medium, spun down, and then resuspended in warm medium. This gets rid of the DMSO, but may disturb fragile cells. If the cells are seeded without spinning, the medium should be changed as soon as the cells have attached.
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from: Bart\'s Cookbook
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.6 d\" _( v; a& G- T# A9 z
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Freezing
1 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. $ X\' P2 A) g4 z2 R& LM
2 Place vials upright in a styrofoam box and cover well. Place in a -70 °C for 24 hours.
3 Place vials in a wand and put in a liquid nitrogen container.

Thawing$ P5 @j- t% r& R
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Preparation
1 Warm water bath to 37 °C. 0 S) e& g\' K6 c5 M: ?# c: v
2 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.
3 Make your growth media for your new culture. For monoclonals this would be DMEM with 20% FBS and penicillin-streptomycin.
Thawing
1 Take your cells out of nitrogen storage and thaw rapidly by swirling in the 37 °C water bath.
2 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.
3 Centrifuge on 3 for 10 min. in a clinical centrifuge. * v/ v\' X5 g\" V+ r
4 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.7 Z. n6 M9 }( V- `- K$ H1 U
from: kitto lab
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2) Determine total cell count of cells to be frozen. (e.g. 1 X 108 )2 ; J\' }8 k. p. f! 5 g
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3) Determine number of vials to be frozen. (e.g. 10 vials at 1 X 107 )# M3 ~) Y; s+ {% V$ h
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4) Centrifuge cell suspension.) _\" y, |3 V6 _0 v) U
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5) Resuspend cell pellet in half the required volume of freezing media. (eg if freezing 10 vials, add 5 mls freezing media); U9 W\" M6 u+ c2 a( E) @. g
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6) Keep cell suspension on ice.
7) Slowly add dropwise with mixing an equal volume of DMSO solution.
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8) Dispense 1 ml of final mixture per sterile Nunc freezing vial.0 H/ G& V3 K* y
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9) Freeze in liquid nitrogen plug or in controlled rate Planar freezer.0 C+ W7 P& v: ?
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Freezing media , K1 i* P6 ~5 Q/ b6 L. X
60 ml RPMI1640 or DMEM
40 ml Gammaglobulin free Horse Serum (or FCS)\" Z- ]# X& I/ B
1 ml Penicillin - Streptomycin\' i* C4 ?- Z+ @2 l
- Filter through 0.2 micro; filter.
- Aliquot in 15 ml conical tubes and store in -200 C freezer for long term periods.. I. R7 |0 c: 9 ; ~|1 l% I
- Aliquots can be stored in tissue culture flasks in refrigerator for short periods.6 `: A# X; O) }) r* `
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20% DMSO solution MAKE FRESH ON DAY OF USE!5 J% K& @# A2 k7 Q4 _- n
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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 micro; filter.( z( Y1 x* p9 Wq
If using 50% DMSO:5 V: i, J8 @$ `. f; c2 ~- I+ M& t
3 ml pH adjusted medium
2 ml 50% DMSO
0.1 ml versene
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- Filter through a 0.2 micro; filter.. N. C2 }9 _1 ^
NOTE: Versene can be left out, but adding it prevents cells from clumping.0 v5 |$ Z2 l0 n1 k$ w0 y7 X\' _5 W* M
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Contributor: Suprya Jayadev9 S# Z, W& K) @B! W) l
Date: January 10, 1991
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