Cell
Screening Service for Protein Secreting Cells Using
The Cell
Screening Service is comprised of five phases: 1) Evaluation of the Cell Line
after encapsulation; 2) Assessment of Reagents; 3) Secretion Assay Optimization;
4) Cell Screening and Selection; and 5) Delivery of the sorted cells.
One Cell Systems works closely with each client to address specific goals
and requirements, and to isolate high secreting cells in a timely,
cost-effective manner.
Length of
Study
Approximately
6-10 weeks, the study includes the following: assay development and
optimization, screening, selection and propagation of cells, and screening of
protein production by supernatant assay. The major factors influencing assay
development are 1) the qualities of the antibody pair used for capture and
detection and 2) the kinetics of protein secretion by each particular cell line.
Reagents
Supplied by Client
Cell
Line
Cryopreserved
cells should be delivered to One Cell Systems on dry ice along with procedures
for initiating cultures, sub-culture and cryopreservation.
If a sub-cloned cell line will be screened, an aliquot of the parental
cell line should also be sent.
Antibody
Pair
A validated,
non-overlapping antibody pair directed against the target protein is required.
Antibody pairs used in a
standard sandwich immunoassay sensitive to 10 pg/mL are usually effective in the
GMD Secretion Assay. Antibodies
should be affinity purified.
The biotinylated
antibody will be used as the capture antibody, which will be attached to the
CelBioGel matrix through a streptavidin bridge.
The other fluorochrome-labeled antibody will be used to detect the
captured secreted protein in GMDs. R-phycoerythrin
(R-PE) or fluorescein isothiocyanate (FITC) are recommended for optimal
detection using flow cytometry.
Protein
Sample
Approximately 1.0
mg of purified protein is necessary to generate assay standards and positive
controls. Alternatively, an aliquot
of the cell culture supernatant with a known concentration of secreted protein
may suffice.
Approved
preferential sera, proprietary (biotin-free) cell culture media and other
reagents specific to the client’s system may also be required.
Cell
Assessment and Assay Development
Evaluation
of the Cell Line
The viability and
growth of cell line of interest after encapsulation will be evaluated.
Compatibility of the cells with surfactants used in the Secretion Assay,
recovery of viable cells from GMDs, and culture methods will also be tested and
optimized. The culture will be
expanded, aliquoted and frozen.
Assessment
of the Reagents
The antibody
pair, protein sample and other reagents will be tested for efficacy in the GMD
Secretion Assay. The target protein
sample, biotinylated capture and fluorochrome-labeled detection antibodies will
be used with unoccupied GMDs to generate a signal titration curve for different
protein concentrations. The
appropriate concentrations of each reagent will be determined empirically to
further optimize the specific GMD Secretion Assay.
Evaluation of alternative antibodies can be performed at additional cost.
Assay
Optimization
In this phase,
optimal conditions will be established for 1) assaying protein secretion from
viable, encapsulated cells, and 2) performing flow sorting of the cell line.
The assay conditions established during the Assay Development Phase will
be used to prepare pilot GMD Secretion Assays.
The assay parameters that result in a strong signal from secreted protein
without saturating GMDs will be determined and validated. To establish good resolution between high and low secretors,
the incubation time, during which protein produced in the GMDs gives a clear
shift above background, will be determined.
Cell
Screening and Selection
Secretion
Assay and Sorting
Using the
conditions established in the assay development and optimization phases, a large
sample will be prepared by encapsulating 3 - 4 aliquots of 1 x 106
cells. The samples will be pooled
and the Secretion Assay performed. The
cells will be sorted using a stringent gate for fluorescence.
High secretors will be selected by sorting those cells exhibiting the
highest fluorescence, approximately 0.5% – 0.1% of the secreting cell
population (at least 1,000 – 2,500 cells).
A typical CHO cell line may exhibit a cloning efficiency of 40%; thus the
1,000 – 2,500 cells collected during the Sorting Phase will result in 400 –
1,000 cells available for further processing.
Additional cells may be isolated from other Secretion Assays of the same
cell line.
Culture
and Screening of the Cells
The selected cells will be cultured for up to three weeks at which point they will be assayed for protein production. Supernatants will be harvested and screened by Gel Microdrop Supernatant Assay.
Cryopreservation
and Delivery of Sorted Cells
Frozen aliquots
of the sorted cells of interest will be delivered at the end of the study.
Procedures for cryopreservation should be provided in advance.
Alternatively, the sorted cells may be stabilized in culture for 24 –
48 hours, after which the flask will be filled with media and the cells shipped
ambient.
Cell Screening Program
Service Agreement
