体外裸鼠内成骨模型实验步骤

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Category by field:Stem cell > Adult stem cell > Mesenchymal stem cell

Category by organism:Mammalia > Murine > Circulatory > Cell-based analysis

In Vivo

Heterotopic Bone Formation Assay

Using Mouse or Human Mesenchymal Stem Cells

Li Chen* and Nicholas Ditzel

Molecular Endocrinology Laboratory (KMEB), Department of Endocrinology, Odense University

Hospital, DK-5000 Odense, Denmark

*corresponding author: Li Chen, lchen@

[Abstract]

Mesenchymal stem cells (MSCs)

are multipotent stromal cells that can differentiate into a

variety of cell types: osteoblasts (bone cells), chondrocytes (cartilage cells), and adipocytes (fat cells).

Seeding

e

xogenous

(human

or

mouse)

MSCs

in

HA

scaffold

and

transplant

subcutaneously

in

immune

-

deficient mice, the cells can finally form bone tissues in the

in vivo

environment.

Detection

and

calculation

the

new

bone

formation

ratios

can

check

the

bone

formation

ability

of

MSCs.

This

protocol describes how to get heterotopic bone formation in mice by mesenchymal stem cells (MSCs)

in hydroxyapatite (HA) scaffolds. This is a simple and robust approach to detect the bone formation by

tissue

engineering

approaches

in vivo

,

and it also

fits

for

examining the roles

of different factors

in

bone formation

with MSCs

.

Materials and Reagents

1.

Human or mouse mesenchymal stem cells (cultured, treated or transfected).

Note:

If

using

the

primary

bone

marrow

stem

cells

(we

took

the

attached

cells

from

bone

marrow culturing), it’s better to use cells in low passages (not over

passage 3); if using MSC

cell lines,

it’s also better to use low passages than high passages.

2.

Mice

[17-Prkdc/J

mice

(NOD/SCID),

around

8

weeks

old]

(The

Jackson

Laboratory)

3.

Hydroxyapatite/tricalcium phosphate (HA/TCP) sca

4.

4% paraformaldehyde

(Sigma, any from local companies should be fine, too)

5.

Hematoxylin/Eosin (H& E) staining solution

(Sigma)

6.

Formic acid solution (see Recipes)

Equipment

1.

1 ml syringes

2.

Cotton sticks (Pro- Ophtha)

3.

Racks (for fixing the syringes in vertical position in cell incubator)

4.

Tools

for

mouse

surgery

(scalpel,

forceps,

needle

holder,

scissors,

sutures,

sterile

swabs,

sterile drapes)

5.

Scan microscopy (Leica

DFC

Microsystems

or any scan microscopy

)

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6.

Computer (with image analysis software, such as ImageJ

or Photoshop CS

)

Software

1.

ImageJ

or Photoshop CS

Procedure

A.

Prepare the implants (

MSCs

with HA/TCP scaffolds)

1.

Take the 1 ml syringes, cut off the tips

with a scalpel and let the syringe tip side open, but

keep the piston inside as it is.

2.

Weight 40 mg hydroxyapatite (HA/TCP) and transfer into each syringe.

3.

Put a cut cotton stick to close the open side of the syringe.

4.

Autoclave the syringes with hydroxyapatite (HA/TCP).

5.

Take the sterilized syringes and HA/TCP back to cell culture room.

Place syringes in a rack

(Syringes must be kept vertical.).

6.

Open the syringes. Add

200

μ

l 10-20% FBS MEM

medium to each HA/TCP scaffold. Mix by 1

ml tip up and down pipetting to wet the HA/TCP thoroughly.

7.

MSC cells

cultured in 10-20% MEM medium

(treated or transfected

with different factors

)

are

trypsinized

and counted.

8.

5 x 10

cells in

200

μ

l 10-20% MEM

medium are transferred into HA/TCP in the syringe.

9.

Mix thoroughly but gently by 1ml tip with up and down pipetting.

10. Place

racks

with

syringes

in

the

incubator

(The

syringes

keep

vertical

straight,

cover

the

syringes by sterilized eppendorf tubes or the cotton sticks), 37

?

C, 5% CO

2

, overnight.

5

?

?

?

?

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11. Next day, bring the transplant cells in the HA/TCP into the animal room. Ready for transplant

cells subcutaneously in mice.

B.

Implantation in mice

1.

NOD/SCID mice, feed in sterilized animal room, around 8 weeks old. Each mouse can hold

four implants in four positions (right-front, left-front, right-back, left-back).

Note:

In

order

to

avoid

variation

induced

by

individual

mice

or

implantation

position,

it

is

suggested

that

in

each

mouse,

with

the

control

and

experiment

group;

in

each

position,

randomly implant the different treated samples.