Expansion of isolated B cells with the B Cell Expansion Kit, human

This application protocol describes the in vitro expansion of primary human B cells.
Many downstream applications require expansion of B cells, due to their low frequencies in peripheral blood. In vitro cultivation of B cells can be time consuming and can often result in dead cells and low expansion rates. The B Cell Expansion Kit, human has a defined formulation that enables standardized expansion of isolated B cells. Expanded cells are fully functional and ready for downstream applications. The expansion rate reaches up to a 10-fold increase after 14 days of in vitro culture. The phenotype and characteristics of the expanded B cells are also comparable to the starting material, therefore preserving all B cell subsets.

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Protocol

1. Things to prepare in advance

Isolation of human PBMCs

For the isolation of human PBMCs refer to the protocol "Isolation of mononuclear cells from human peripheral blood by density gradient centrifugation".

Reagent preparation

Preparation of PEB buffer (for staining of isolated B cells)

PEB buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA by diluting MACS® BSA Stock Solution (# 130-091-376) 1:20 with autoMACS® Rinsing Solution (# 130-091-222). Keep buffer cold (2–8 °C). Always use freshly prepared buffer.
Do not use autoMACS Running Buffer or MACSQuant® Running Buffer as they contain a small amount of sodium azide that could affect the results.

2. Separation of B cells

For B cell isolation various reagents and kits are available according to the kind of isolation.

Untouched isolation of B cells from PBMCs by depleting all unwanted cells.
- Pan B Cell Isolation Kit, human (# 130-101-638)
- Memory B Cell Isolation Kit, human (# 130-093-546)
B cell isolation directly from blood products using StraightFrom® MicroBeads.
That allows magnetic cell separation directly from human blood products, such as whole blood, buffy coat, leukoreduction system chambers (LRSCs) and leukopaks.
- StraightFrom® Whole Blood CD19 MicroBeads, human (# 130-090-880)
Label-free B cell isolation.
REAlease® Technology allows for magnetic cell isolation with subsequent removal of any beads and labels from cells of interest. Clever utilization of recombinant antibody fragments allows for cell surface labeling in just a few steps.
- REAlease® CD19 MicroBead Kit, human (# 130-117-034)
B cell isolation.
For the protocol B cells were separated using MACS® MicroBeads. For details refer to the respective data sheet.
- CD19 MicroBeads, human (# 130-050-301)

Download the quick guide and find a comprehensive overview of all B cell markers and products for convenient isolation and analysis.

3. Staining of isolated B cells

Labelling of B cells

To obtain the results shown in this protocol a total of 1×10⁶ cells has been stained in a total volume of
100 µL.

Centrifuge cell suspension at 300×g for 10 minutes. Aspirate supernatant completely.
Prepare one master mix with all fluorochrome-conjugated antibodies according to the respective data sheets.
Add one master mix per tube containing cell suspension.
Mix well and incubate for 10 minutes in the dark in the refrigerator (2−8 °C).
▲Note: Higher temperatures and/or longer incubation times may lead to non-specific cell labeling. Working on ice requires increased incubation times.
Wash cells in each tube by adding 1 mL of PEB buffer and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
Resuspend cells in an appropriate volume of PEB buffer (1× PBS, 0.5% BSA, 2 mM EDTA) and proceed to flow cytometry analysis.

Flow cytometry analysis

Prior acquisition of samples in a flow cytometer, e.g., the MACSQuant® Analyzer 10, make sure lasers are properly calibrated (by using, e.g., the MACSQuant Calibration Beads), and compensation of the spillover of fluorochrome emissions has been established in advance using cells or the MACS® Comp Bead Kit, anti-REA. Prior acquisition adjust also forward scatter (FSC)/side scatter (SSC) parameters to properly locate all leukocyte populations in the plots.

Exclude doublets by using the FSC-H/FSC-A parameters.
Use gated single cells to exclude erythrocytes and debris using the SSC/FSC parameters.
Use the SSC/FSC gate to exclude dead cells from the analysis using the 7-AAD Staining Solution.
Use gated viable cells to identify B cell subsets.

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Figure 1: Flow cytometric analysis of B cells.

4. B cell culture

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Figure 2: General concept of B Cell Expansion Kit, human with IL-4. Cross-linking of CD40 expressed on B cells leads to cell activation and proliferation. The B Cell Expansion Kit consisting of a recombinant CD40L multimer, a cross-linking antibody, and IL-4. Combining CD40L together with IL-4, which is a known growth factor for activated B cells, has been shown to be crucial for effective activation and proliferation of B cells.

Reconstitution of lyophilized reagents

Reconstitute lyophilized Human CD40-Ligand (component of the B Cell Expansion Kit, human) with deionized sterile-filtered water to a final concentration of 0.5 mg/mL in a volume of 200 μL (for 100 μg size) or 1000 μL (for 500 μg size) and store at –20 °C.
Reconstitute human IL-4 with deionized sterile-filtered water to a final concentration of 2.5×10⁵ IU/mL, e.g. if the lot has a biological activity of 8×10⁶ IU/mg, 160 μL water should be used for reconstitution of 5 μg.
Mix components by pipetting up and down until resuspended. To avoid repeated freeze-thaw cycles, reconstituted reagents should be aliquoted and stored at –20 °C or below until use.

B cell expansion

This protocol is optimized for the activation and expansion of B cells, using the Human CD40-Ligand Multimer Kit (component of the B Cell Expansion Kit, human). B cells can either be expanded in low density (0.15×10⁶ cells/mL) or high density (1×10⁶ cells/mL) cell culture.

Volumes given below are for 10 mL of final expansion medium. When working with larger volumes please scale up accordingly.
The final expansion medium should be prepared directly before the stimulation. Long-term storage is not recommended.
▲Note: In case you want to differentiate and expand B cells towards a matured, switched status use Human IL-21, premium grade at a concentration of 0.7 U/mL instead of human IL-4.

To increase the biological activity, Human CD40-Ligand and Cross-Linking Antibody (component of the B Cell Expansion Kit, human) must be pre-incubated. Mix 40 μL reconstituted Human CD40-Ligand with 40 μL Cross-Linking Antibody. Incubate for 30 minutes at room temperature (19–25 °C).
Prepare 10 mL StemMACS™ HSC Expansion Media XF (component of the B Cell Expansion Kit, human) with
2 µL reconstituted Human IL-4, and 5% AB serum. Add the multimerized Human CD40-Ligand (80 μL) to prepared media to get the final expansion medium.
Resuspend B cells at a density of 1×10⁶ cells/mL or 0.15×10⁶ cells/mL of the final expansion medium. Culture in an appropriate plate (for details refer to table 1).
Incubate at 37 °C, 5% CO₂.
On days 7 and 10 exchange half of the medium with fresh medium supplemented with 5% AB serum and
50 units IL-4/mL (0.2 μL human IL-4 per mL media from step 2). Harvested supernatants can be used for downstream analysis.
On day 14 harvest B cells and proceed to downstream applications.

Table 1: Culture plate sizes and amount of medium for different cell sizes.

Total cell number (high density)	Total cell number (low density)	Final medium volume	Culture plate
0.5×10⁶	0.075×10⁶	0.5 mL	48 well
1.0×10⁶	0.15×10⁶	1 mL	24 well
2.0×10⁶	0.3×10⁶	2 mL	12 well
4.0×10⁶	0.6×10⁶	4 mL	6 well

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Figure 3: Expansion of B cells. To facilitate expansion, B cells were isolated on day 0 from human buffy coat using CD19 MicroBeads, human after performing a density gradient. The B cells were cultured using the B Cell Expansion Kit. Therefore, components of the CD40-Ligand Multimer Kit were pre‑incubated for 30 minutes at room temperature. Afterwards, 80 μL multimerized Human CD40-Ligand were added to 10 mL prepared StemMACS HSC Expansion Media XF and added to the isolated B cells. B cells were expanded with a starting density of 0.15×10⁶ cells/mL (low density) according to the protocol. On day 7 and 10 cells were harvested, counted, and restimulated. All cells were harvested, counted, and analyzed on day 14. With this protocol, a B cell expansion of up to 10-fold was achieved. These experiments were performed in three independent experimental replicates.

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Figure 4: Subset distribution of expanded B cells. To determine the phenotype of isolated B cells and differentiation on day 14 of culture, a gating strategy was developed for analysis of cells by flow cytometry. After debris and doublets were excluded, viable CD19⁺ cells were selected and analyzed for the proportions of naive and memory cells according to their CD27 and IgD expression. Viable plasma cells were determined as CD138⁺CD38⁺⁺ cells. The amounts of naive B cells decreased under all culture conditions on day 14, as naive B cells have a shorter survival rate. In contrast, the amounts of non-switched memory B cells increased in both the unstimulated culture and the culture expanded by the B Cell Expansion Kit and IL-4 indicating a greater survival capacity of this subset. Interestingly, B cells cultured with the kit and IL-4 seemed to resemble untreated B cells in their subset distribution, while B cells cultured with the kit and IL-21 (instead of IL-4) varied from cells cultured under the other conditions. In the presence of IL-21, a significant increase in the plasma cell fraction (up to 20% of all B cells) was observed while switched memory B cells doubled from 22 to 45%. This suggests that the culturing condition provided by the B Cell Expansion Kit plus IL-21 drives B cell differentiation towards a matured, switched status. These experiments were performed in three independent experimental replicates.

Materials

The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. These products are for research use only.

Reagent and instrument requirements

Isolation of PBMCs using Ficoll-Paque™

PBS/EDTA: Phosphate-buffered saline (PBS), pH 7.2, with 2 mM EDTA.
▲Note: EDTA can be replaced by other supplements, such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD).

Ficoll-Paque™ (ρ=1.077g/mL)

50 mL conical centrifuge tubes

Buffer

autoMACS® Rinsing Solution (# 130-091-222)

Bovine serum albumin (MACS® BSA Stock Solution # 130-091-376)

PEB buffer: PBS, pH 7.2, 0.5% BSA, and 2 mM EDTA

Magnetic cell separation

CD19 MicroBeads, human (# 130-050-301) or Pan B Cell Isolation Kit, human (# 130-101-638)

LS Columns (# 130-042-303)

MACS® Separator for LS Columns, e.g. MidiMACS™ Separator (# 130-042-302)

MACS MultiStand (# 130-042-303)

Pre-Separation Filters (30 µm) (# 130-041-407)

B cell activation and expansion

B Cell Expansion Kit, human (# 130-106-196)

Human IL-21, premium grade (# 130-095-784), 0.7 U/mL

Analysis of isolated B cells

Antibodies, human

Marker	Clone	Dye	Order no.
CD138	44F9	PE	# 130-119-840
CD38	REA671	FITC	n/a
CD27	REA499	PE-Vio® 770	# 130-113-641
IgD	REA740	VioBlue®	# 130-110-786
CD20	LT20	VioGreen™	# 130-113-379
CD19	REA675	APC-Vio® 770	# 130-113-643
CD24	REA675	APC	# 130-113-642
(Optional) Propidium Iodide Solution or 7-AAD Staining Solution			# 130-111-568

MACSQuant® Analyzer 10 (# 130-096-343) or other flow cytometers equipped with violet (405 nm) and red (635 nm) lasers able to discriminate FITC, PE, and APC fluorescence.

MACS Chill 96 Rack (# 130-094-459) when using MACSQuant Analyzer 10

MACSQuant Calibration Beads (# 130-093-607) when using MACSQuant Analyzer 10

MACS Comp Bead Kit, anti-REA (# 130-104-693) for optimal compensation of the fluorescence spillover from fluorochrome-conjugated antibodies

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