sp00212-the hybridoma workflow is improved by ......fusion #1 fusion #2 fusion #1 fusion #2 fusion...

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The standard workflow for generating hybridoma cell lines is inefficient and time-consuming. B cells isolated from lymphoid tissue or blood of an immunized animal are fused to myeloma cells, followed by selection and screening to identify hybridomas secreting antigen-specific antibodies. Typically, the desired hybridomas are then expanded, followed by several rounds of limiting dilution cloning to achieve monoclonality and verify stable antibody production by the selected clones. Improvements to the upstream (pre-fusion) and downstream (cloning/expansion) steps of the hybridoma workflow have the potential to substantially increase the efficiency while reducing the burden, time and cost associated with this widely-used technique. Here we report the use of immunomagnetic enrichment and expansion of B cells prior to fusion to significantly improve antigen-specific hit rates and fusion efficiencies, respectively, compared to non-enriched or non-cultured cells. We also report development of a serum-free, animal origin-free (AOF) hybridoma culture medium that provides limiting dilution cloning efficiencies similar to or higher than a commonly used growth medium (DMEM + 10% fetal serum) and facilitates purification of the desired antibody free of contaminating serum-derived immunoglobulins. Together, these reagents offer improvements at three critical steps in the hybridoma workflow.

Introduction

Protocol

SummaryEasySep™ immunomagnetic selection of CD138+ cells prior to fusion significantly improves the hit rate for hybridomas expressing antigen-specific antibodies and simplifies the screening process.

Culturing splenocytes or immunomagnetically-isolated B cells in a serum-free (xeno-free) medium prior to performing fusions substantially enhanced the hybridoma fusion efficiency.

An animal origin-free medium (ClonaCell™-HY AOF Expansion & Cloning Medium) has been developed that supports robust cloning and expansion of hybridoma cells in the absence of serum at the very low plating densities used during single-cell cloning.

The Hybridoma Workflow is Improved by Incorporating CD138+ Cell Selection, B Cell Activation and Expansion, and Serum-Free CultureAlexis Maxwell1, Tracy Lee1, Trevor Rogers1, Whalley Fong1, Sandra Babic1, Yunyun Shen1, Steven Woodside1, Terry Thomas1, Allen Eaves1,2, and Mark Brown1

1STEMCELL Technologies Inc., Vancouver B.C., Canada 2Terry Fox Laboratory, BC Cancer Agency, Vancouver BC, Canada

Results

ClonaCellTM-HY Medium B

ClonaCellTM-HY Medium C

ClonaCellTMHY PEG

Pre-Fusion & Cell Isolation Expansion & Cloning Growth, Ab ProductionFusion Selection

ClonaCellTM-HY Liquid HAT Selection Medium

ClonaCellTM-HY Medium D(Semi-Solid Selection)

ClonaCellTM-HY Medium Dwithout HAT (Semi-Solid Selection)

ClonaCellTM-HY Medium E

ClonaCellTM-HY AOF Expansion & Cloning Medium

NEW



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ClonaCellTM-HY Medium A

EasySepTM Mouse CD138 Positive Selection Kit

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B Cell Activation & Expansion Supplement

NEW


NEW

FIGURE 1. The Efficiency of Hybridoma Generation can be Improved by Incorporating B Cell Isolation, B Cell Expansion, and Use of a Serum-Free (Animal Origin-Free) Medium at Key Steps within the Workflow.

Fusion Efficiency

TABLE 2. Fusion Efficiency is Increased by Culturing Cells in B Cell Activation and Expansion Medium.Total splenocytes, Pan-B cells and CD138+ cells were isolated from immunized mice and fused to Sp2/0 myeloma cells either on the day of cell isolation (Day 0) or after being cultured in Base Medium with or without B Cell Activation and Expansion Supplement for 2 – 4 days prior to fusion. For each fusion ~5 x 105 total splenocytes or cultured cells were mixed with an equivalent number of myeloma cells. Hybridomas were selected in HAT-containing semi-solid medium with ClonaCell™-HY Hybridoma Kit (Catalog #03800) for 14 days, after which the colonies were counted and the fusion efficiency (# colonies / # splenocytes) was determined and normalized to the total splenocyte (non-cultured) control.

FIGURE 7. Cloning Efficiencies for Three Hybridoma Cell Lines Subcloned in Serum-Containing and Serum-Free Cell Culture Media. The hybridoma lines were adapted to growth in each medium and subcloned by limiting dilution. After incubation for 10 days (37°C, 5% CO2) the plates were analyzed with a Cell Metric™ instrument (Solentim). The plating efficiency was estimated by Poisson statistics using the ELDA method described by Hu & Smith, 2009 (J Immunol Meth, 347: 70-78). ACF = animal component-free. Data is expressed as mean + 1SD.

FIGURE 8. Expansion of an Established Hybridoma Cell Line in Several Commercially-Available Serum-Containing and Serum-Free Hybridoma Cell Culture Media. The hybridoma line was adapted to growth in each medium and seeded in triplicate in 24-well tissue culture plates at 5 x 103 cells/mL. The cultures were incubated at 37°C (5% CO2 atmosphere) and the viable cell density was measured on days 3 – 7. Data is expressed as mean ± 1SD.

FIGURE 3. Antigen-Specific Hit Rates for Hybridomas Generated from EasySepTM-Isolated CD138+ Cells or Total Splenocytes. Isolated CD138+ cells or total splenocytes from immunized BALB/c mice were fused with Sp2/0 mouse myeloma cells and plated in semi-solid medium using the ClonaCell™-HY Hybridoma kit (Catalog #03800). Hybridoma colonies were picked into liquid medium for expansion and the percentage of antigen-specific (circles) and antibody-secreting (triangles) hybridomas determined by ELISA. Data is expressed as mean ± 1SD.

FIGURE 4. Expansion of Mouse B Cells Cultured in Base Medium with or without B Cell Activation and Expansion Supplement.B cells were enriched from splenocytes of naïve or immunized mice using EasySep™ Mouse Pan-B Cell Isolation Kit (Catalog #19844) or EasySep™ Mouse CD138 Positive Selection Kit (Catalog #18957; coming soon) and cultured in either Base Medium or Base Medium containing B cell Activation and Expansion Supplement. Cells were cultured for 4 – 11 days and the viable cell concentration was determined at various time points by flow cytometry. The observed maximum fold expansion of viable cells is shown as mean ± 1SD.

Isolated CD138+

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FIGURE 5. In Vitro Induction of Differentiation of Mouse Pan-B Cells with B Cell Activation and Expansion Supplement.Pan-B cells were enriched from splenocytes of a naïve mouse using EasySep™ Mouse Pan-B Cell Isolation Kit (Catalog #19844) and cultured in medium with and without B Cell Activation and Expansion Supplement. Expression of CD138 and CD267 (TACI), which are markers highly expressed by plasma cells/blasts, was analyzed by flow cytometry at day 0 (A), day 3 (B, D) and day 6 (C, E). Differentiation to plasma cells was also accompanied by upregulation of the activation marker CD86, increased antibody production, and a pronounced increase in the Forward-Scatter profile of the cells (consistent with the larger size of plasma cells relative to unactivated B cells) (data not shown).

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Serum-Containing: DMEM + 10% FBS, ClonaCellTM-HY Medium E Serum-Free (with Serum-Derived Proteins): Medium 1

Serum-Free: Medium 2, 3, 4Animal Component-Free: Medium 5Animal Origin-Free: ClonaCellTM-HY AOF MediumProtein-Free: Medium 7, 9Chemically-Defined: Medium 11, 12, 13

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FIGURE 2. Representative Flow Cytometry Data for Isolation of CD138+ Cells from Splenocytes of Immunized Mice using EasySep™ Immunomagnetic Positive Selection. CD138+ cells were enriched from splenocytes of an immunized BALB/c mouse using EasySep™ Mouse CD138 Positive Selection Kit (Catalog #18957; coming soon). The viable CD138+/CD267 (TACI)+ (plasma cell/blast) cell content is shown for (A) start cells (total splenocytes), and (B) isolated cells. Similar results were obtained using EasySep™ immunomagnetic positive selection of rat CD138+ lymphocytes (data available upon request).

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TABLE 1. Purities and Average Cell Yields of Viable Mouse CD138+ Cells Isolated by Immunomagnetic Positive Selection with EasySep™. Purity values are expressed as mean ± 1SD and cell yield is indicated as the average number of cells recovered per 1 x 108 start cells (splenocytes). Results were obtained using EasySep™ Mouse CD138 Positive Selection Kit (Catalog #18957; coming soon) to process spleen samples from naïve C57BL/6 or immunized BALB/c mice and include data for EasySep™ Magnet, "The Big Easy" Magnet, and EasyEights™ Magnet.

Purity Target cells recovered per

1 x 108 start cellsNaïve Mouse

Spleen 52 6.0 ± 2.1 41 75.0 ± 10.2 0.36 x 106

Immunized Mouse Spleen

43 5.9 ± 2.3 4 77.9 ± 1.6 2.48 x 106

CD138 Total

SampleStart Isolated

n n(% ± SD)

Purity(% ± SD)

Purity Target cells recovered per

start cellsNaïve Mouse

Spleen 52 0.4 ± 0.1 41 49.5 ± 16.2 0.24 x 106

Immunized Mouse Spleen

43 1.6 ± 1.1 4 71.6 ± 3.4 1.47 x 106

Plasma cells/Plasmablasts

SampleStart Isolated

n n(% ± SD)

Purity(% ± SD) 1 x 108

Fusion #1 Fusion #2 Fusion #1 Fusion #2 Fusion #1 Fusion #2Total

Splenocytes1.00 1.00 4.44 0.35 27.33 36.62

Pan B 0.47 7.56 18.67

CD138+ Isolated Cells

2.22 0.63 36.76

Cells Cultured in Base Medium (Day 2 or 4) Cells Non-cultured cells (Day 0)

(Normalized to Non-cultured Total Splenocytes)Cells Cultured in

Base Medium With Supplement (Day 2 or 4)

Isolated Cells

Fusion Efficiency

Pan-B cells (naive mice) Pan-B cells (immunized mice) CD138+ cells (immunized mice)

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FIGURE 6: Cloning Efficiencies for Ten Hybridoma Cell Lines Subcloned in Serum-Containing and Serum-Free/Animal Origin-Free Cell Culture Media.Hybridoma clones secreting antibodies specific for a variety of antigens were subcloned by limiting dilution in either DMEM containing 10% FBS, the serum-containing ClonaCell™-HY Medium E, or the serum-free (animal origin-free; AOF) ClonaCell™-HY AOF Expansion & Cloning Medium (Catalog #03835; coming soon). After incubation for 12 - 14 days (37°C, 5% CO2) the plates were examined under a microscope and assessed for growth. The cloning efficiency was estimated by Poisson statistics using the ELDA method described by Hu & Smith, 2009 (J Immunol Meth, 347: 70-78). Insert (B) shows the average cloning efficiency obtained for the ten hybridoma cell lines in each cell culture medium. Data is expressed as mean + 1SD.

* Cloning efficiencies of 0% were observed in 18 separate experiments for Polymer 2, and in 1 experiment for Protein 4.

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sp00212-the hybridoma workflow is improved by ......fusion #1 fusion #2 fusion #1 fusion #2 fusion...

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