现象英语细胞培养Cell_culture_technology

Cell Culture Technology

Summary

Cell culture is an invaluable tool for investigators in numerous fields. It facilitates analysis of

biological

properties

and

processes

that

are

not

readily

accessible

at

the

level

of

the

intact

organism. Successful maintenance of the cell culture, whether primary or immortalized, requires

knowledge and practice of a few essential techniques. The purpose of this paper is to explain the

basic principle of the cell culture using the maintenance of the adherent primary cell line.

The first necessity is a well-established and properly equipped cell culture facility. The level of

biocontainment required (Level 1-4) is dependent on the type of the cells cultured and the risk that

these cells might contain and transmit infectious agents. For example, culture of the primate cells,

transformed

human

cell

lines,

mycoplasma-contaminated

cell

lines,

and

nontested

human

cells

require a minimum of a Level 2 containment facility. All facilities should be

equipped with the

following: a certified biological safety cabinet that protects both the cells in culture and the worker

from biological contaminants; a centrifuge, preferably capable of refrigeration and the equipped

with

appropriate

containment

holders

that

is

dedicated

for

cell

culture

use;

a

microscope

for

examination of cell cultures and for counting cells; and a humidified incubator set at 37

℃

with

5% CO

2

in air. A 37

℃

water bath filled with water containing inhibitor (

化抑制剂

,

缓蚀剂

,

抑制

者

)

of bacterial and fungal growing can also be useful if warming of media prior to use is desired.

Although these are the basic requirements, there are numerous considerations regarding location

of the facility, airflow, and other design features that will facilitate (

促 进

)

contamination-free(

污

染

;

沾染

)

culture. If a new cell culture facility is being established, the oper should consult (

会诊

,

咨

询

)

facility

requirements

and

laboratory

safety

guidelines

that

are

available

from

your

，

institution

s biosafety department or the appropriate government agencies.

The

second

requirement

for

successful

cell

culture

is

the

practice

of

the

sterile(

消毒方法

)

technique.

Prior

to

beginning

any

work,

the

biological

safety

cabinet

should

be

turned

on

and

allowed

to

run

for

at

least

15

min

to

purge

(

净化

< br>,

清除

)

the

contaminated

air.

All

wor

k

surfaces

within

the

cabinet

should

be

decontaminated

去污

with

an

appropriate

solution;7

0%

ethanol

乙醇

or

isopropanol

are

routinely

used

for

this

purpose.

Any

materials

required

for

the

procedure

操作

should

be

similarly

decontaminated

and

placed

in

or

near

the

cabi

net.

This

is

especially

important

if

solutions

have

been

warmed

in

a

water

bath

prior

to

u

se.

The

oper

should

don

appropriate

personnel

protective

equipment

for

the

cell

type

in

q

uestion.

Typically

,

this

consists

of

a

lab

coat

with

the

cuffs

袖口

of

the

sleeves

secured

with

masking

tape

遮蔽胶带

to

prevent

the

travel

of

biological

contaminant

and

Latex

or

vinyl

gloves

that

cover

all

exposed

skin

that

enters

the

biosafety

生物安全

cabine

t.

Gloved

hands

should

be

sprayed

喷雾

with

decontaminant

prior

to

putting

them

into

the

cabinet

and

gloves

should

be

changed

regularly

if

something

outside

the

cabinet

is

touche

d.

Care

should

be

taken

to

ensure

that

anything

coming

in

contact

with

the

cells

of

interest,

or

the

reagents

needed

to

culture

and

passage

them,

is

sterile

消毒的

(either

auto

claved

or

filter-sterilized).

The

biosafety

office

associated

with

your

institution

is

a

valuabl

e

resource

for

providing

references

related

to

the

discussion

of

required

and

appropriate

te

chniques

required

for

the

types

of

cells

you

intend

to

use.

A

third

necessity

for

successful

cell

culture

is

appropriate,quality

controlled

reagents

and

supplies. There are numerous suppliers of tissue culture media (both basic and specialized) and

supplements.

Examples

include

Invitrogen

(),Sigma-Aldrich

(

),

BioWhittaker

(),and

StemCell

Technologies

Inc.().

Unless

otherwise

specified

in

the

protocols

草案

accompanying

your

cells

of

interest,

any

source

of

tissue- culture-grade

reagents

should

be

acceptable

for

most

cell

culture purposes. Similarly, there are numerous suppliers of the plasticware needed for most cell

culture applications (i,e.,culture dishes and/or flasks, tubes, disposable pipets). Sources for these

supplies

include

Corning

(/lifesciences/),

Nunc

(),

and

Falcon (/discovery-labware). Two cautionary

警戒的

notes are essential.

First,

sterile

无菌培养

culture

dishes

can

be

purchased

购买

as

either

tissue

culture

treated

or

Petri style. Adherence cells require tissue-culture-treated dishes for proper adherent and growth.

Second, it is possible to use glassware rather than disposable

一次用弃的

plastic for cell culture

purposes.

However,

it

is

essential

that

all

residual

cleaning

detergent

is

removed

and

that

appropriate sterilization (i.e.,121

℃

for at least 15 min in an autoclave

高压锅

) is carried out prior

to the three above-listed requirements have been satisfied, the final necessity for successful

cell culture is the knowledge and practice of the fundamental techniques involved in the growth of

the cell type of interest. The purpose of this chapter is to explain the basic principles of cell culture

using

the

maintenance

of

an

adherent

cell

line

the

BHK-21

cell

as

examples.

Procedures

for

resuscitation of frozen cells;

growth and maintenance of live cell cultures; flask

细胞培

养瓶

usage; changing media; splitting

分裂

cell cultures

and freezing cell cultures

are

described.

Cell Culture Standard Protocol

Resuscitation

复苏

of frozen cells:

1.

Place a pack of the appropriate media in a 37

℃

water

bath to warm about ? hour

before removing ampoule from dry ice.

2. Leave ampoule at room temperature for about 1 minute. Transfer to a 37C water bath

for

1-2

minutes

until

fully

thawed

解冻的

.

Quickly

thawing

融化

the

ampoule

will

minimise any damage to the cell membranes. Be careful not to totally immerse

浸入

the

ampoule

–

this may increase contamination

污染

risk.

3. Wipe

擦

ampoule with a tissue soaked in 70% alcohol prior to opening.

4.

Slowly

pipette

the

whole

ampoule

into

flask

containing

pre-warmed

medium.

A

small (25 cm) flask with 5 mL of medium will be a good start for the cells.

5. Incubate in tissue culture CO2 incubator.

Growth and maintenance of live cell cultures:

1.

Check

on

cells

at

least

every

other

day.

Inspect

cells

macroscopically

and

with

microscopic viewer in cell culture room. Observe for obvious colour change indicative

of possible contamination (change from pink to yellow) or less drastic colour change

due

to

build-up

of

waste

products

in

media.

Any

contaminated

flasks

should

be

discarded immediately. Change media in viable flasks every couple

双

of days.