Biomedical Engineering Reference
In-Depth Information
SWNTs.and.the.streptavidin.in.the.endosomes.(detected.by.luorescent.labeling).indicated.
an.uptake.pathway.consistent.with.endocytosis.
15.3.3.2 Cell Selection and Screening Techniques
Screening.and.identiication.of.desired.populations.of.cells.is.currently.a.daunting.chal-
lenge..Whether.the.cell.lines.are.hybridomas.that.must.be.selected.for.antibodies.with.high.
afinity.to.the.antigen.of.interest.or.CHO.or.microbial.cells.in.which.high.productivity.is.
desired,.current.methods.are.laborious.and.time.consuming..Typically.mammalian.cells.
are.plated.in.96-well.plates.using.serial.dilution.to.obtain.single.cell.clones..Clones.are.then.
screened.for.antibody.afinity.or.productivity.and.then.cell.clones.of.interest.are.sequen-
tially.scaled.up.to.larger.culture.volumes.to.verify.productivity.in.stirred.or.shaken.con-
ditions..Similar.approaches.are.taken.for.microbial.populations.with.the.exception.being.
that.colonies.are.typically.isolated.from.semi-solid.media.(e.g.,.agar)..The.introduction.of.
luorescence-activated.cell.sorting.has.improved.the.rate.of.cell.screening.to.some.degree,.
but.it.is.typically.used.to.increase.the.number.of.cells.scanned.rather.than.to.reduce.overall.
amount.of.labor.expended..The.same.is.true.for.automated/robotic.culture.methods..They.
have. been. used. to. increase. clone. screening. from. hundreds. to. thousands.. A. novel,. very.
creative.alternative.approach.has.been.proposed.by.Love.and.coworkers.(Love.et.al..2006,.
2010;.Ogunniyi.et.al..2009;.Ronan.et.al..2009;.Choi.et.al..2010;.Gong.et.al..2010;.Love.2010;.
Panagiotou.et.al..2011)..In.a.2006.
Nature Biotechnology
.paper.(Love.et.al..2006),.they.describe.
a.micro-engraving.method.in.which.an.array.of.microwells.50.mm.in.diameter.and.depth.
(separated.by.50.mm).or.100.mm.(separated.by.100.mm).was.fabricated.in.PDMS.(0.1-1.nL.
total. volume).. The. array. was. designed. to. it. within. the. boundaries. of. a. 1.in..×.3.in.. glass.
slide,. a. common. format. for. microarrays.. After. sterilization. with. an. oxygen. plasma. and.
coating.of.the.wells.with.BSA,.hybridoma.cells.were.then.deposited.on.the.microwell.array.
at.a.concentration.that.would.allow.one.to.three.cells.in.∼50%.to.75%.of.the.wells..After.
removing.the.excess.cells.and.media,.the.microwell.array.was.then.placed.in.contact.with.
an.assay.slide.(see.Figure.15.8).on.which.either.the.antigen.of.interest.or.a.capture.antibody.
had.been.placed..The.binding.of.antibodies.produced.by.the.cells.in.the.microwells.could.
then. be. “read. out”. by. treating. the. glass. slide. with. an. appropriate. luorescently. tagged.
antibody..They.subsequently.used.this.technique.to.screen.newly.formed.hybridoma.cells,.
identifying. 4,300. positive. cells. from.∼200,000. cells. screened. by. the. microarray. technol-
ogy..This.technology.permits.the.screening.of.up.to.∼80,000.cells.per.plate.with.antibody.
detection. possible. in. as. little. as. 2.h. after. plating,. an. enormous. increase. in. throughput..
Subsequent.studies.have.extended.this.work.to.screening.of.high.productivity.clones.of.
Pichia pastoris
.(Love.et.al..2010;.Panagiotou.et.al..2011).and.have.incorporated.automation.
for.improved.single.cell.recovery.(Choi.et.al..2010).
In.a.somewhat.different.approach,.several.groups.have.applied.porous.aluminum.oxide.
(PAO). as. a. substrate. for. development. of. micro-. or. nanoarrays. for. culture. and. assay. of.
mammalian.cells.and.microorganisms..Ingham.et.al..(2007).irst.described.a.micro-Petri.
dish.for.microbial.growth..In.this.report,.reactive.ion.etching.(RIE),.a.process.of.etching.
by.using.plasma.directed.by.shadow.mask.(14),.was.used.to.selectively.remove.an.acrylic.
polymer. laminated. on. the. surface. of. the. PAO. to. create. areas. open. for. growth.. They.
were.able.to.create.surfaces.>340,000.compartments.per.cm
2
..The.smallest.compartment.
size. made. was. 7.×.7.μm. at. the. base,. which. gave. 1. million. discrete. growth. areas. on. an.
8.×.36.mm. chip.. This. density. is. comparable. to. 40. million. compartments. on. the. footprint.
of. a. standard. multiwell. plate.. The. most. commonly. used. compartment. size. (8.×.36.mm.
chip,. 20.×.20.μm
−1
. growth. areas,. 20.μm-wide. walls). had. 180,000. compartments. per. chip..
