protocol-humen stem cell culture

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Culture of Human Embryonic Stem Cells (hESC)
8 f. H* i7 S. |: P- e; U& y  YAll cell lines are initially grown according to the supplier's protocols but we are adapting them to one simple protocol outlined below:' j$ r: `! u) a$ s* ~8 h

* ]% f  y4 C3 e' B! G, B# L6-well plates (Falcon Cat #353046) are coated for 20 to 60 minutes at room temperature with 0.1% gelatin (Sigma Cat #G1890) in dH2O. / H5 c( H; I  L2 u) j0 p4 X7 c
Mouse embryonic fibroblasts (CF1 strain), cultured in MEF medium, are mitotically inactivated by treatment with 10μg/ml mitomycin C (Roche Cat #107 409) for 2 to 3 hours at 37°C. Cells are washed three to four times with PBS, trypsinized (Invitrogen Cat #25300-054), and plated at a density of 0.75 x 105/ml with 2.5ml per well of a gelatin-coated 6-well dish. Alternatively, cells may be inactivated by exposure to 8000rads of X-irradiation and plated at the same density. : h5 E* k' a" a. h4 @+ o3 o2 A
Immediately before plating hESC, MEFs are rinsed once or twice with PBS. hESC are plated onto MEFs as small clumps in 2.5ml per well of hESC medium containing 4ng/ml bFGF (R&D Systems Cat #233-FB). Cells are fed every day until ready to passage which is determined by the size of colonies, the age of MEFs (should not be older than 2 weeks) or differentiation status of the cells. * J( h" K/ {7 ~7 r
Colonies which appear to be differentiating, are manually removed before passaging. ( d9 j* `- Y) G8 C% d0 ?/ H
To passage hESC, cells are washed once or twice with PBS and incubated with filter-sterilized 1mg/ml collagenase IV (Invitrogen Cat #17104-019) in DMEM/F12 for 10 to 30 minutes. Plates should be agitated every 10 minutes until colonies begin to detach. When moderate tapping of the plate causes the colonies to dislodge, they are collected and the wells washed with hESC medium to collect any remaining hESC. Alternatively, colonies may be removed using a cell scraper and collected.
: ]# R8 J7 L1 C2 e1 N6 }Colonies are allowed to sediment for 5 to 10 minutes. The supernatant, containing residual MEFs, is aspirated, and the colonies are washed with 5ml hESC medium and allowed to sediment again. This is repeated once more.
: E! \0 m# }) a/ c9 [, A% FAfter the final sedimentation, the colonies are resuspended in 1ml of hES medium and triturated gently to break up the colonies to approximately 100-cell size. Generally, cell lines are passaged at a ratio of between 1:3 and 1:6 every four to seven days. 2 |7 D+ r7 i/ U
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" ~) \( @9 U6 b& b# m# GMEF Medium DMEM Invitrogen 11965-092 450ml & ?4 g3 }$ |9 Z
Heat-inactivated FBS Invitrogen 16000-044 50ml
# q! {  e6 P: B7 i, R9 ^Non-essential amino acids Invitrogen 11140-050 5ml
' r& q0 @. ~% o% mL-Glutamine Invitrogen 25030-081 5ml
6 D! V5 N* o4 v- r7 S; [3 fhESC Medium DMEM/F12 Invitrogen 11330-032 400ml
2 O( b0 u4 g' f8 E# KKnockout Serum Replacer Invitrogen 10828-028 100ml 5 P! R; ]; ^/ Y( b1 j4 u8 O8 P! Y
Non-essential amino acids Invitrogen 11140-050 5ml # J  r0 ~# b6 p1 P) R: ^
L-Glutamine Invitrogen 25030-081 2.5ml , `. a% O& O. ]7 ]) N
β-mercaptoethanol Sigma 7522 3.5μl
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$ d0 a  U# e# h/ ?from:http://stemcells.nih.gov/research/NIHresearch/scunit/culture.asp

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Culturing BG01V Human Embryonic Stem Cells with Mouse Embryonic Fibroblast (MEF)-Conditioned Media- R0 v2 l. p# e& p- Y
If culturing in the absence of a feeder cell layer is desired, human embryonic stem (hES) cells can be maintained using Mouse or Human-Conditioned Media (Catalog # AR005, AR007). The protocol below has been used with the BG01V line of hES cells. Please note that other hES cell lines may require modifications of this protocol. Optimal culture conditions must be determined by the investigator for each hES line.1 f" G2 M" z1 q2 p
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Please read the protocol in its entirety before starting.
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Supplies Required! o# y0 D3 |6 p4 }2 i
Reagents) b) I5 w& h& H! Y; N4 H: I% C
MEF-Conditioned Media (R&D Systems, Catalog # AR005) or Human Feeder Cell-Conditioned Media (R&D Systems, Catalog # AR007)
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Recombinant human FGF basic (R&D Systems, Catalog # 233-FB) or tissue culture grade FGF basic (R&D Systems, Catalog # 4114-TC)
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8 d# E/ T$ H6 ^- i! m  SAccutase (Innovative Cell Technologies, Catalog # AT104 or equivalent)! A* L% H: f: [8 W9 p; M! n

1 U1 f& _/ d  vCultrex® Reduced Growth Factor Basement Membrane Extract (BME) (R&D Systems, Catalog # 3433-005-01)
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DMEM/F12
0 d4 M$ z, v5 {3 rMaterials& Q4 t2 c3 e) b8 g  l7 c
BG01V human embryonic stem cells (ATCC, Catalog # SCRC-2002)
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60 or 100 mm tissue culture plates   @" G1 D, U" i1 P8 j1 J  }2 f7 G
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15 mL centrifuge tubes% T& g9 a0 b1 {' R" K  o

2 Y, _  V/ U6 [Pipettes and pipette tips
/ `. p: W: u" W# O" E4 o0 wEquipment
0 |4 |* Q2 o9 P! T- H37° C, 5% CO2 incubator
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) _2 W& B- P2 W2 _2 MCentrifuge (low speed clinical or equivalent)
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Hemocytometer+ g5 E3 G/ W4 |' l/ V0 Z6 W- Q
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Microscope
% P0 S' [" a: O0 \% b2 ~- m' [2 YFigure 1
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Procedures
/ s& L" z  A  i* @3 R! xNote: When handling biohazard materials such as human cells, safe laboratory procedures should be followed and protective clothing should be worn./ C, Q  \+ p& G

+ K8 i! O- J# L  I& KNote: Sterile technique is required when handling the reagents.
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1 |. d$ q& w$ T+ R7 A/ F% APrepare the Cultrex BME-coated plate. (Figure 1)
5 Z. Q% f' B+ `% t& S) CThaw Cultrex BME on ice at 2 - 8° C overnight. ' r' _3 E: M  u+ u5 F( U4 H. J
Aliquot thawed Cultrex BME into pre-cooled tubes and store at -20° C. ) w& o5 Z1 i0 ~% X6 T7 ^
Thaw the aliquot on ice or at 2 - 8° C overnight. 5 o* Y! i' q: y1 J4 c$ N
Dilute Cultrex BME 1:40 in DMEM/F12. This can be stored at 4° C for up to 2 weeks.
& w& U. P6 B' _5 K7 l+ P1 p, S- e: w/ _Coat the desired number of plates with diluted Cultrex BME (approximately 2.5 mL/60 mm plate) and incubate for 1-2 hours at room temperature.
+ V! |! v/ }* P% K1 WRemove the Cultrex BME solution immediately prior to plating the cells.
% f. j2 X% {1 RPreparation and plating of BG01V Cells. (Figure 2) Figure 26 {  e" R- G  V& \2 n
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Warm the MEF or Human Feeder Cell-Conditioned Media to 37° C.
, n& ^: a/ j. v* r. f; v  @+ n  A0 }Warm the vial of BG01V hES cells until just thawed and then immediately transfer to a 15 mL centrifuge tube containing at least 5 mL of pre-warmed Conditioned Media. Rinse the cryovial with an additional 1 mL of media to ensure the removal of all the cells.
( S6 a% G. X) ^; O8 QSpin in a clinical centrifuge at 200 x g for 4 minutes.
( z+ B- h$ [0 ORemove the supernatant and gently flick the pellet. Resuspend the pellet in an appropriate amount of Conditioned Media supplemented with 4 ng/mL of recombinant human FGF basic.
& G! p& b! s4 h8 p) x$ e4 vAdd the BG01V hES cell suspension to the Cultrex BME-coated plate. / g% _2 i0 H4 N! b- \
Grow in a 37° C, 5% CO2 incubator. Change the media daily and monitor the cells. Passage the cells at the desired confluency.
% C4 S1 Q. J! L- f- v! R. L5 W& p& BPassaging BG01V Cells (Figure 3)
& v6 k( D% X( z: x. B. @Prepare the desired number of plates by coating with Cultrex BME, as described above, 1 - 2 hours prior to passaging the cells.
' Q( ^; t' X/ u, d; X4 F; M6 }: F1 eWarm the MEF- or Human Feeder Cell-Conditioned Media to 37° C.
7 C4 s" D, ]1 A, G' v4 q5 p$ zRemove the Conditioned Media from cells. Add 1 mL of Accutase solution to each 60 mm plate. Incubate at room temperature for 5 - 10 minutes or until cells begin to slough off the plate.
, A3 `' Y7 W5 Z0 o7 y$ N" s  U/ ^Pipette gently over the plate until all the cells have been detached. 5 S# `3 P! o* w/ D. s) }2 ^
Pipette the cell suspension up and down to break up large cell clumps. ) r8 D* E  i8 v5 j# U
Remove the cell suspension to a 15 mL centrifuge tube containing 5 mL of Conditioned Media and spin at 200 x g for 4 minutes.
  B9 s3 E% R9 N' c% I1 V& w" ZResuspend the pellet in Conditioned Media and count the viable cells using a hemocytometer.
$ h% Y  f9 t8 L  t0 u* \Plate the desired number of cells (approximately 1.0 x 106 cells/60 mm plate) on the Cultrex BME coated plate in Conditioned Media containing 4 ng/mL of recombinant human FGF basic.
8 N. x' D3 a$ u, o& `Change the media daily. Monitor the cells for the desired confluency.' F( e* d+ }  I: f1 w8 S! q* H# C

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BG01V cells are licensed from BresaGen, Inc.
8 n# H5 x/ @' t2 F: C. \" XCultrex is a registered trademark of Trevigen, Inc.

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