生物分离工程 课件-双语 Chapter 8 Magnetic Separation

Chapter9Magnetic

Separation“Upgrade”T

cellTheTopTenScientificAdvancesof2013CAR-T

Cell

TherapyCAR-T细胞疗法：嵌合抗原受体T细胞免疫疗法ChimericAntigenReceptorT-CellImmunoterapyPrimary

Task:How

to

get

the

specific

T

cellsMagneticSeparationCAR-T

Cell

Therapy肿瘤免疫治疗第二章离心第五章膜分离第四章沉淀第七章离子交换、凝胶过滤、亲和层析Why

do

we

need

another

separation

methods?第八章磁分离CAR-T

Cell

Therapy5

1.Whatistheprincipleandcompositionofmagneticseparation?2.Why

do

we

use

magneticseparationtopurifyspecificTcells

rather

than

chromatography

or

other

methods?3.Whatarethelimitationsofmagneticseparation?Outline1.

Principle2.

Formation3.

Magnetic

Separation

Process4.

Application

&

DiscussionPrincipleTraditionalmethodsforbio-separationinvolvetheuseofvariouschromatography

and

membrane

separation,howeverthesemethodsrequiretheuseofmultipleexpensiveinstrumentsandarelaborious.Magneticbioseparationisdistinctfromthebioseparationmethodsrelyingonexternalforcesofgravity,centrifugalforces,orelectricforces,asitisbasedontheeffectofamagneticforces.Principle磁分离原理BindingthetargettoamagneticsubstancethroughspecificinteractionsMagnetic

Separation磁性物质磁性物质PrincipleAdvantages

of

Magnetic

SeparationThetargetmaterialcanbeconvenientlyandquicklyseparatedfromacrudesuspensioncontainingsolidparticles.Whenseparatingintracellularproteins,thecelllysisandproteinseparationstepscanbecombinedtoreducetheseparationtime.Itcanbeusedtoconcentratethesampleforfurtherchromatographyorelectrophoresisanalysis.Theshearforcesduringtheelutionprocessarethesmallestcomparedtocentrifugationorfiltration,andtheappliedstaticmagneticfieldwillnotaffecttheionsandsolutesinthesolution(atlowflowrates).Inconclusion,magneticseparationcangentlyandquicklyseparatebioactivesubstancesfromaliquidenvironmentthatisnotconducivetotheirexistence.Principle11

Howtoobtainmagneticbeadswithcoatedaffinityligands?磁分离原理PrincipleCouplingligandstothesurfaceofmagneticbeadstoachievespecificandreversiblebindingofproteinsandmagneticseparationthroughaffinityinteractions.Magneticmicrospheresarecomposedoftwoparts:nanometer-scale(1~100nm)superparamagneticinorganicnanomaterials(Fe,Co,Niandtheiroxides,etc.)andpolymers.Core-shelltype[includingmagneticcoretypeandmagneticshelltype],mixedtypeandmultilayertype.磁性材料聚合物Formation13TargetAffinityLigandsMagnetic

BeadsMBs磁分离过程Formation14TargetAffinityLigandsSteric

HindranceMagnetic

BeadsMBs磁分离过程Formation15TargetAffinityLigandLinkerMagnetic

BeadsMBs磁分离过程FormationTheprotein

solutionismixedwithmagneticbeads.Theprotein-binding

magneticisattractedbythemagneticseparator,thesupernatantisdiscardedorusedforotherpurposes.Thecomplexiswashedmultipletimestoremoveimpurities.Theproteinsareelutedfromthemagneticbead.MagneticSeparationProcessMagneticSeparationProcess18Separation

of

T

CellsHow

to

separate

T

cells？CD3

markerT

cell

receptor淋巴细胞T细胞CD3

antibodySeparation

of

T

Cells20MagneticBeadsMagneticBeads2HNSepharoseMagneticBeads磁珠活化Separation

of

T

Cells淋巴细胞CD3T

cellMBs—CD3

antibodypH=7.4BindingpH=3.5ElutionT

cellsDiscardOther

cellsSeparation

of

T

CellsMagnet-ActivatedCellSorting

(MACS)Separation

of

T

CellsCan

you

proposeaT

cellpurificationprocess?Can

you

proposeaT

cellpurificationprocess?CollectionCentrifugationGradient

CentrifugationMagnetic

Separation

(MACS)FCM

AnalysisEngineering从实验对象中采集外周血低速离心分离细胞Ficoll梯度离心法分离淋巴细胞偶联CD3抗体的磁珠分离，酸洗脱改造T细胞流式细胞术检测T细胞纯度23Separation

of

T

Cells辅助T细胞细胞毒性T细胞CD3

markerT

cell

receptorCD3

markerT

cell

receptorHow

to

separate

cytotoxic

T

cells？Separation

of

T

CellsThelpercellsCytotoxicTcellsMagneticBeadsCD3antibodyMagneticBeadsCD8antibody+Helper

T

cells

CD4

positiveCytotoxic

T

cellsCD8

positiveMBs—CD8

antibodypH=7.4BindingpH=3.5ElutionDiscardHelper

T

cellsCytotoxicTcellsSeparation

of

T

CellsCD3

markerT

cell

receptorCD3

markerT

cell

receptorCD3

markerT

cell

receptor辅助T细胞细胞毒性T细胞调节性T细胞Separation

of

T

CellsThelpercellsCytotoxicTcellsRegulatoryTcellsHow

to

further

separate

cytotoxic

T

cells

and

remove

the

Tregs？MagneticBeads+MagneticBeadsMagneticBeads+CD3antibodyCD8antibodyCD25antibodyCD8CD25pH=7.4BindingCytotoxic

T

cellsCD8

positiveCD25

negativeSeparation

of

T

CellsCan

you

proposeacytotoxic

T

cellpurificationprocess?CollectionCentrifugationGradient

Centrifugation1st

MACS2nd

MACS3rd

MACSFCM

AnalysisEngineering从实验对象中采集外周血低速离心分离细胞Ficoll梯度离心法分离淋巴细胞偶联CD3抗体的磁珠分离，酸洗脱改造细胞毒性T细胞流式细胞术检测细胞毒性T细胞纯度28Separation

of

T

Cells偶联CD8抗体的磁珠分离，酸洗脱偶联CD25抗体的磁珠分离，收集上清液Separation

of

T

CellsDiscussionChallenges：Tcellsarenotsufficientlyactivated.Acidelutionhaslowefficiency,leadingtopoorcellrecoveryandreducedactivity.Multipleroundsofmagneticseparationcausedamagetothecells.Optimized

Separation

ProcessCollectionCentrifugationGradient

Centrifugation1st

MACSActivation2nd

MACS3nd

MACS…从实验对象中采集外周血低速离心分离细胞Ficoll梯度离心法分离淋巴细胞偶联CD3抗体的磁珠分离总T细胞，化学断键or酶切释放质控、改造、扩增、纯化偶联CD25抗体的磁珠去除Tregs31加入抗体、细胞因子活化T细胞偶联CD8抗体的磁珠分离细胞毒性T细胞FACS

separationFluorescence-Activated

CellSorting

(FACS)

canresolvetheissueofcelldamagebyenablingprecise,single-stepsortingwithouttheneedformultipleseparationrounds.免疫磁分离Separation

of

T

Cells