animal cell culture -...
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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
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7
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Preparation of Cell Banking
• This works if you remember:
– Two stocks: M & W (master & working cell bank)
– Store cultures appropriately (cells & document)
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Effect of freezing rate – with cryoprotection
Cells dehydrate but many survive
Slow cooling
+ cryoprotectant
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Effect of freezing rate – with cryoprotection
Cells dehydrate but many survive
Fast cooling
Slow cooling
Death from ice crystal damage
+ cryoprotectant
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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
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How to do it - Step 1 (Master stock)
Harvest, pool
and freeze
down 20 vials
No antibiotics!
(master stock,
passage:N)
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How to do it - Step 2 (expansion)
Master stock
Thaw 1 vial and
check recovery
Expansion
No antibiotics!
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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
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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
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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
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Controlling the freezing rate
Best – electronically
controlled cooling
• Very reliable
• Expensive
• High capacity
• Permanent record
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Cryogenic storage systems
3 approaches:
Mechanical freezers
(-135° to -150°C)
Submerged in LN2 (-196°C)
In vapor phase LN2 (-140 to 196°C)
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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