Measurement of transfection efficiency Fluorescent proteins

minerbendBiotechnology

Feb 12, 2013 (4 years and 6 months ago)

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Flow Cytometry and Cell Sorting for Biotechnology Institute for Applied Microbiology, Vienna
Cell Line Optimisation and Transfection
1
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
Measurement of transfection efficiency

Typically done using a
reporter protein

Enzymes:

ß-galactosidase

Live cell permeable substrate

Nonfluorescent

Chemically modified by ß-gal
to become fluorescent

Fluorescent proteins

Fluorescent per se

Derived from different marine
organisms

Enhanced forms with
humanised codons and
optimised genetic elements

Mutants with enhanced
fluorescence and with
different wavelengths
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
Fluorescent proteins

Blue fluorescent proteins

EBFG: ex/em 390/445 nm

Sapphire: ex/em 399/511 nm

Cyan fluorescent protein

ECFP: ex/em 440/480 nm

Green fluorescent protein

Ex/em480/510 nm

Yellow fluorescent protein

Ex/em 514/530

Red and Orange FP

mOrange ex/em 548/562

dTomato ex/em554/581

DsRed ex/em558/583

mCherry ex/em587/610

mPlum ex/em590/650

Often used as fusion proteins
for confocal microscopy
Flow Cytometry and Cell Sorting for Biotechnology Institute for Applied Microbiology, Vienna
Cell Line Optimisation and Transfection
2
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
Transfection experiment

Parameters: % transfected
and level of expressed
protein

Red: negative control

Green: 1µg DNA = 52%

Blue: 2 µg DNA = 69%

Orange: 5 µg DNA = 82%
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology

Dihydrofolate Reductase

co-transfected with genes of
interest

integrate at same chromosomal
site with a certain frequency

required: a dhfr- cell line which will
grow in the presence of nucleotide
precursors (HAT)

amplification of gene copy number
by stepwise increase of
methotrexate concentration
Degree of Amplification
Folate
Tetrahydrofolate
DHFR
MTX(Folatanalogon)
binds to and
inhibits DHFR
Purin- and Thymidin-
Biosynthesis
Flow Cytometry and Cell Sorting for Biotechnology Institute for Applied Microbiology, Vienna
Cell Line Optimisation and Transfection
3
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology

MTX-FITC

passage cells at least twice
without MTX to remove
intracellular traces

incubate for 18h with MTX-
FITC

analyse
Degree of Amplification

Expression of dihydrofolate
reductase does not
necessarily correlate to
expression of product
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
Sorting of Producer Cells

Intracellular measurements
require cell fixation

methods to permeabilise
membranes to allow specific
antibodies to pass (f.i.
electroporation) reduce
viability

ER retention vs. Secretion??

Dhfr amplification:

not necesarrily correlated

potential?

Viable Methods

Surface expression

Microencapsulation

Cold Capture

Secretion Assay
Flow Cytometry and Cell Sorting for Biotechnology Institute for Applied Microbiology, Vienna
Cell Line Optimisation and Transfection
4
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
Surface Expression

usually only found in
Hyridoma cells

normal developmental feature
of lymphocytes

different pathway of gene
regulation and synthesis

causes stimulation of B-cells
producing a specific antibody

initiates antibody maturation

present on some hybridoma
cells

does not necessarily
correlate to secretion rate

if confirmed: USE

but never directly after a
fusion

make sure to eliminate dead
cells
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
Microencapsulation

developed by James Weaver,
MIT, One Cell Systems

encloses cells in soft agarose
coupled to biotin

a capturing antibody is bound
via an avidin bridge

secreted product is bound by
the antibody and retained in
the capsule
Flow Cytometry and Cell Sorting for Biotechnology Institute for Applied Microbiology, Vienna
Cell Line Optimisation and Transfection
5
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
Microencapsulation

easily adapted

should work with most cells
and products

many applications also for
microbiology, growth
assays...

special equipment required

larger nozzle size

recover cells from agarose

licence
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
Cold Capture

Brezinsky et al., (Biogen) JIM, 2003, 277, p141

putting cells on ice will leave some of the secreted product
sticking in the membrane or the glycocalyx

staining with a labelled antibody

Sorting

Simple, fast and inexpensive method

Use of possible
Flow Cytometry and Cell Sorting for Biotechnology Institute for Applied Microbiology, Vienna
Cell Line Optimisation and Transfection
6
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
Single Cell Affinity Matrix Secretion Assay

Manz et.al (1994), Proc. Nat. Acad. Sci. 92: 1921

Affinity Matrix on Cell Surface

catches secreted product on cell surface
of
live
cells

anchored via biotin-avidin bridge

highly flexible method, both with regard
to cell lines and different products

incubation in high viscosity medium to
reduce diffusion and carry over

secreted product stained with
fluorescent antibody
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
Affinity matrix

Biotin Anchor

NHS-succinimidyl-Biotin

water-soluble variant

may reduce viability and
cloning efficiency at higher
concentrations

biotinylated surface ligand

anything that binds to a
protein present on the cell
surface without influencing
activity or stimulation

f.i. antibody to CD45 for
hybridomas or lectins
binding to carbohydrate
residues

Check viability, Cloning Efficiency and potential effects on
stimulation
Flow Cytometry and Cell Sorting for Biotechnology Institute for Applied Microbiology, Vienna
Cell Line Optimisation and Transfection
7
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology

succinylated-Concanavalin A-
Biotin

succinylation prevents
oligomerisation of
concanavalinA molecules,
thus reducing the biological
activity

ConA binds to mannose
residues, present on all
glycosylated surface proteins

can also be used on yeast
cells

optimal concentration for
CHO and hybridoma cells:
40-50µg/ml

may have to be optimised for
other cell lines

determination of binding with
avidin-FITC (or other label)

check influence on viability
and cloning efficiency
Affinity matrix
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology

Catching and Staining
Antibody

test suitability of antibody
pair in ELISA and then with
known producer cell line

polyclonal antibodies may
work better

commercial availability?

special considerations

f.i. in recombinant antibody
producing cell lines

ratio of light to heavy chain

catcher: anti-heavy chain

staining antibody: anti-light
chain
Affinity matrix
Flow Cytometry and Cell Sorting for Biotechnology Institute for Applied Microbiology, Vienna
Cell Line Optimisation and Transfection
8
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
wide range of specific production rates
10
20
30
40
50
60
70
80
0 10 20 30 40
Qp [pg.cell-1.day-1]
[relative units]
30 min
20 min
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
Incubation

To prevent cross-contamination
of non-producing cells with
antibody secreted by other cells
and not „caught“ on the cell
surface immediately

Incubation in high-viscosity
medium containing

25% gelatine
cell culture tested, dialysed

3-5% Carboxy-methyl-
cellulose in PBS, autoclaved
Flow Cytometry and Cell Sorting for Biotechnology Institute for Applied Microbiology, Vienna
Cell Line Optimisation and Transfection
9
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
Sorting for high production rates
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
Dead cells
Flow Cytometry and Cell Sorting for Biotechnology Institute for Applied Microbiology, Vienna
Cell Line Optimisation and Transfection
10
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
Sorting for high production rates
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
Amplification of recombinant Chinese Hamster
Ovary Cells

human monoclonal antibody

Co-Transfection of the genes for heavy and light
chain with dehydrofolate reductase

amplification by stepwise increase in
Methotrexate concentration
Flow Cytometry and Cell Sorting for Biotechnology Institute for Applied Microbiology, Vienna
Cell Line Optimisation and Transfection
11
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
Staining Design

CHO cells may produce heavy or light chains alone

selection of antibodies

anti-heavy chain for catching

anti-light chain for staining
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
Isolation of High Producing Subclones

limited dilution cloning: 45 plates, 1500 wells tested

sorting: 5 plates, 19 wells tested
0
10
20
30
40
50
60
70
80
0 1 2 3 4 5 6 7 8 9 10
MTX concentration [ µM]
Qp [pg.cell
-1
.day
-1
]
Flow Cytometry and Cell Sorting for Biotechnology Institute for Applied Microbiology, Vienna
Cell Line Optimisation and Transfection
12
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
Clone Homogeneity
neg. control 0,8µM MTX
3,2µM MTX 6,4µM MTX 9,6µM MTX1,6µM MTX
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
Sort Design and additional cell properties

complete antibody molecules already included in staining
protocol

cell robustness included in procedure

stability in the absence of selective pressure

appropriate production kinetics

growth rate, serumfree growth.........
Flow Cytometry and Cell Sorting for Biotechnology Institute for Applied Microbiology, Vienna
Cell Line Optimisation and Transfection
13
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
Isolation of stable subclones
0
5
10
15
20
25
0 50 100 150 200 250 300 350 400
TIME [days]
qP [µg/106cells.day]
control + MTX
control - MTX
sort 1
sort 2
sort 3
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
Isolation of stable subclones
0
10
20
30
40
50
60
70
80
90
100
no MTX with MTX Sort 1 (2) sort 2 (5) sort 3 (5)
Percent of initial qP
after 90 days
after >120 days
Flow Cytometry and Cell Sorting for Biotechnology Institute for Applied Microbiology, Vienna
Cell Line Optimisation and Transfection
14
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology

Sort Goal: cells with high production rates at density inhibition

Sort design: sort 3-5 days after confluence is reached
Changing production kinetics of an
established cell line
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
Changing production kinetics of an
established cell line
Red Line: Cell density 0.5x 10
5
/cm
2
Green Line: Cell density 1x 10
5
/cm
2
Blue Line: Cell density 2x 10
5
/cm
2
Orange Line: Cell density 3x 10
5
/cm
2
Flow Cytometry and Cell Sorting for Biotechnology Institute for Applied Microbiology, Vienna
Cell Line Optimisation and Transfection
15
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
0
5
10
15
20
25
30
35
40
0 50000 100000 150000 200000 250000 300000 350000
Cell Density [cells/cm2]
specific secretion rate [rel.Units]
Comparison of secretion rates of
original clone
and
sorted
clones
at different cell densities
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
Batch cultures
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5 6 7 8
Time [days]
specific productivity
[pg/cell/day]
Increase in qP, when
medium is replaced on
day 7:
+15%
+75%
+165%
Flow Cytometry and Cell Sorting for Biotechnology Institute for Applied Microbiology, Vienna
Cell Line Optimisation and Transfection
16
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
Single Cell Secretion Assay
Sort/2H6
Sort/2F7
Red Line: Cell density 0.5x 10
5
/cm
2
Green Line: Cell density 1x 10
5
/cm
2
Blue Line: Cell density 2x 10
5
/cm
2
Orange Line: Cell density 3x 10
5
/cm
2
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
Present Problems in Hybridoma
Screening

very time, labour and material consuming

specific production rate not as high as desired

no time and resources left to screen

for specific properties of the antibody

for other cellular properties but specific production rate
Flow Cytometry and Cell Sorting for Biotechnology Institute for Applied Microbiology, Vienna
Cell Line Optimisation and Transfection
17
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
State of the Art

limited dilution cloning

2-5% IgG-producers

of these less than 5% specific

for 1 specific mAb 400-1000 wells have to be tested

3 to 10 rounds of subcloning

to ensure monoclonality

to find high producing subclones
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
Hybridoma Cell Line Isolation

8-12 days after fusion sort for
IgG production at 5-10 cells/well

sort 2-4 plates, freeze remaining
cells

check titer for IgG and
specificity

select polyclonal populations
producing specific antibodies

sort for specificity at 2 cells/well

again sort 1-4 plates, freeze
remaining cells

check titer for ratio of total
IgG/specific IgG

finally sort for high production
rates
Flow Cytometry and Cell Sorting for Biotechnology Institute for Applied Microbiology, Vienna
Cell Line Optimisation and Transfection
18
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
Possible Applications

Sorting of IgG-producer Cells after Fusion
maximum 10 cells per well, spec. production rates between 10 and 25 pg/cell/day
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
Rescue of unstable cell lines
0
2
4
6
8
10
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140
Time [days]
qAB [µg huIgG/10E6cells.day]
Parental clone
Subclone 2
Subclone 1
SORT
Flow Cytometry and Cell Sorting for Biotechnology Institute for Applied Microbiology, Vienna
Cell Line Optimisation and Transfection
19
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
Antigen specificity
• Direct staining of product with antigen:
• further decrease in work load
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
Antigen Specificity

Double Staining with anti-
mouse IgG-PE or APC or QuR
and an FITC conjugated
antigen

if antigen is tagged (f.i. 6-His)
use FITC-labelled anti-His-Tag
antibody

after secretion phase incubate
for 30 min on ice with antigen

wash

incubate with anti-His-tag-ab
and anti-m-IgG-ab
Flow Cytometry and Cell Sorting for Biotechnology Institute for Applied Microbiology, Vienna
Cell Line Optimisation and Transfection
20
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
To optimise cell performance

Characterize cell behaviour

decide on desirable cell
properties

find staining combinations
that characterize this property

Sort under conditions that
reflect those in „real life“
Universität für Bodenkultur Vienna Depar tment of Biotechnology Institute of Applied Microbiology
Library screening

Numerous system described:

Baculovirus/insect cells

Staphylococcus

Yeast cells

Mammalian cells

For reasonable efficiency requires a high speed sorter

Analysis of 10e8 cells

Normal sorter (1000 cells/sec) takes 26h

High speed sorter (30000 cells/sec) takes less than 1h

Screening for

Ligand binding

Interaction

Mostly by surface display