Animal Cell Culture

孫立易

自我簡介

孫立易 (Sun, Li-Yi)

學歷：

• 國立交通大學 生物科技系 博士 (2004/09/01~2010/09/15)

• 國立東華大學 生物技術研究所 碩士 (2002/09/01~2004/06/30)

• 國立東華大學 生命科學系 學士 (1997/09/01~2001/06/30)

現任：

• 慈濟綜合醫院 研究部 基因既幹細胞研製中心 博士後研究員(2011/07/04~)

曾任：

• 台灣大學高分子科學與工程學研究所 博士後研究員(2011/05/01~2011/6/30)

• 拜寧奈米生醫股份有限公司 實驗室主管 (2009/10/01~2011/04/30)

• 拜寧奈米生醫股份有限公司 專案室主管 (2010/09/16~2011/04/30)

• 天賜爾生物科技股份有限公司 助理研究員 (2001/08/01~2002/08/31)

專長：

• 體細胞製劑GTP製程開發

• 成體幹細胞體外大量化製程

• 幹細胞核心技術

• 生物反應器原理及實作

• 藥物測試之細胞模型建立

• ISO17025測試實驗室品質系統

動物細胞種類

• 動 物 細 胞可依其性質、形態及培養特性來分類。

• 依性質而分， 有初代細胞(primary cul t u r e ) 、正常細胞(normal cell ) 及轉型細胞( t ransformed cell)三種。

• 初代細胞是直接來自動物組織的細胞， 通常取得方式是由切碎的組織中加上蛋白酶分解而得。

• 正常細胞是指具雙倍染色體， 有一定的存活期限， 且無法永續培養的細胞。轉型細胞則沒有存活限制， 細胞可持續培養。

• 部分轉型細胞的生長並不需要附著面， 細胞密度也不受限制， 染色體亦有變化。

• 體 外 培 養 細胞若以其基本形態區分， 可分為成纖維型細胞、上皮型細胞、游走型細胞及多形態型細胞等四種。

一般平面培養

6

7

11

Preparation of Cell Banking

• This works if you remember:

– Two stocks: M & W (master & working cell bank)

– Store cultures appropriately (cells & document)

12

Effect of freezing rate – with cryoprotection

Cells dehydrate but many survive

Slow cooling

+ cryoprotectant

13

Effect of freezing rate – with cryoprotection

Cells dehydrate but many survive

Fast cooling

Slow cooling

Death from ice crystal damage

+ cryoprotectant

14

Key steps for freezing cells

• Start with healthy, actively growing cells

• Use slow freezing to prevent intracellular ice crystals

• Use cryoprotectants to minimize dehydration effects

• Store cells below -130°C to stop biological time

15

How to do it - Step 1 (Master stock)

Harvest, pool

and freeze

down 20 vials

No antibiotics!

(master stock,

passage:N)

16

How to do it - Step 2 (expansion)

Master stock

Thaw 1 vial and

check recovery

Expansion

No antibiotics!

17

How to do it - Step 3 (Working stock)

Harvest, pool

and freeze

down 20 vials

(Working stock,

passage:N+2) Cells from

seed stock

p:N

+2

p:N

+2

p

:N+

2

p:N

+2

18

How to do it - Step 4 (check)

Harvest, pool

and freeze

down 20 vials

Cells from

seed stock

Thaw I vial, check viability

and test for contamination

Once tested, a new vial is

used every 3 months or 10p

working stock

Selecting a storage vessel:

Plastic vials:

• Several cap designs with or without o-rings or gaskets

• 1 to 5mL sizes

• Easy to label

• May leak in liquid nitrogen – can explode!

• Accessories

20

Controlling the freezing rate

OK - Do-It-yourself cooling using foam boxes

• Needs 12 - 24 hr at -80°C

• Often works but has higher degree of variability

• Low cost

• Large space

• No record

Controlling the freezing rate

Good –

• Needs overnoght at -80°C

• Reliable

• Medi-capacity

• Relatively inexpensive (NT 2000-4000)

• Reusable

• No record

Mr. Frosty

Coolcell

22

Controlling the freezing rate

Best – electronically

controlled cooling

• Very reliable

• Expensive

• High capacity

• Permanent record

23

Cryogenic storage systems

3 approaches:

Mechanical freezers

(-135° to -150°C)

Submerged in LN2 (-196°C)

In vapor phase LN2 (-140 to 196°C)

24

2 types of LN2 storage

In Vapor

Phase

In Liquid

Phase

• Lower than -140°C

• Very safe

• Shorter holding time

• Reduced vial capacity

• -196°C

• Longer holding time

• Greater vial capacity

•Danger of vials exploding!

• Contamination issues!

動物細胞的大量培養應用 • 動物細胞的大量培養應用，最初是因病毒的生產而起。

• 1949 年Ender 發現小兒麻痺病毒可以在體外細胞中培養應用，奠下疫苗製造的基礎。

• 1950 年代小兒麻痺疫苗開始大量接種， 同時也是第一個上市的動物細胞製品。

• 之後其他產品陸續地開發， 大量培養與生產條件的探討， 成為動物細胞培養技術的研究重點。

• 1987 年第一個利用遺傳工程動物細胞生產的藥用蛋白質tissue plasmiongen activator 核准上市。

Roller Bottle Culture

The cell monolayer is

constantly bathed in

liquid, but is

submerged only for

about one- fourth of

the cycle, enabling

free gas exchange

Roller Culture Bottle on Racks

Small benchtop rack Large benchtop or free-standing

extendable rack

Examples of Roller Culture Bottles

Center and lift, disposable plastic (Falcon, Corning);

right, glass

Nunc Cell Factory

Filling Nunc Cell Factory

Run the medium in. Rotate the system

onto its short side

away from the

inlet.

Lay and seal the

inlet, or connect the

inlet to a 5﹪-CO2

line.

Roller Drum Apparatus

Roller drums are

used for roller

culture of large

numbers of small

bottles or tubes.

Large Stirrer Flask Stirrer Culture

Biostat

Air-Lift Fermentor

5﹪CO2 in air is pumped into a

porous steel ring at the base of the

central cylinder, and bubbles

stream up the center, carrying a

flow of liquid with them, and are

released at the top, while the

medium is recycled to the bottom of

the cylinder.

Rotating Chamber Systems

High-molecular-weight products remain with

cells and can be harvested from the sampling

ports, while replenishment of the medium is

carried out via the right-hand ports. Mixing is

achieved by rotating the chamber on a roller

rack.

The techne system relies on enclosing the

cells in angled, permeable tubes, with an air

bubble located in each tube. As the culture

rotates, the bubble moves up and down the

tubes, ensuring mixing.

Hollow-Fiber Perfusion

Sectional diagram of a hollow-fiber

perfusion system. Medium is

circulated from a reservoir to the

culture chamber by a peristaltic

pump. Aeration and CO2 exchange

are regulated in the reservoir of

medium.

NASA Bioreactor

Cells are maintained in the

central chamber, rotating at a

speed such that they do not

sediment.

Microcarrier Culture

Microcarriers

Fixed- and Fluidized-Bed Reactors

The beads are usually glass and

are settle in a dense bed resting on

a perforated base at the bottom of

the culture vessel. Once the culture

is established, the beads do not

move, and medium percolates

around the them.

Beads of a lower density are used

and float on the low-velocity

perfusate

Bioreactor Process Control