3DProSeed StromaLine Bone-Marrow Vascular Niche

Contents and Storage

• StromaLine Bone-Marrow MSC (part no. ECT.STRL.MSC): 1x vial >750'000 of cryopreserved human cryopreserved multipotent stromal cells isolated from normal (non-diabetic) adult human bone marrow to be stored in liquid nitrogen.

• StromaLine Bone-Marrow EC (part. no. ECT.STRL.BMEC): 1x vial 1'000'000 of cryopreserved endothelial cells isolated from the iliac crests of normal human donors; immortalized by transfection with the SV40 virus and stably expressing EGFP under the CMV promoter and puromycin as the selection marker.

• StromaLine Bone-Marrow MSC Medium (part no. ECT.STRL.MSC_M): 1x 500mL complete medium optimized for the growth of the StromaLine bone marrow MSC and Vascular Niche in 3DProSeed hydrogels. Store at 2-4 °C.

• StromaLine Bone-Marrow EC Medium (part no. ECT.STRL.BMEC_M): 1x 500mL complete medium optimized for the growth of the StromaLine Bone-Marrow Vascular Niche.

• Stroma Line Assay Microtiter Plate (part no. ECT.PSSTRL): 1x 3DProSeed 96-well glass-bottom hydrogel plate optimized for the growth of the StromaLine bone marrow Vascular Niche. Store at RT.

Product Overview

The StromaLine Bone-Marrow Vascular Niche is an optimized platform for generating 3D co-cultures of mesenchymal stromal cells (MSC) and endothelial cells (EC) isolated from normal adult human bone marrow on a synthetic PEG-based hydrogel that allows the deposition and assembly of a native extracellular matrix resembling the stromal component of the bone-marrow vascular niche. The hydrogel is optically transparent, allowing a wide range of microscopy-based assays. Additionally, the hydrogel can be enzymatically dissolved at the end-point of the culture, and the cells, as well as the extracellular matrix fraction, can be retrieved and processed for further biochemical analysis, including proteomics and transcriptomics analyses. The hydrogel is offered pre-casted and ready to use in a 96-well plate format, and the cells can be delivered directly into your laboratory pre-plated, growing in the hydrogel plate or cryopreserved and ready for seeding in the plate.
The StromaLine cells, medium, and hydrogel plate are quality tested together and guaranteed to give optimum performance as a complete system.

Seeding Protocol for Cryopreserved Cells

Establishment of the MSC supporting mono-culture​

1.    Thaw the StromaLine Bone-Marrow MSC Medium (delivered frozen) at 37 °C for upon arrival or prior to use. Do not refreeze. Prepare 50-mL aliquots and store at 4 °C for up to 1 month. Avoid repeating cycles of warming as this results in reduced activity of the growth factors used to supplement the medium and suboptimal culture growth. 
2.    Bring the StromaLine Assay Microtiter Plate at room temperature and the StromaLine Bone-Marrow MSC Medium aliquot to be used at 37 °C for at least 30 min prior to use.
3.    Thaw the frozen StromaLine Bone-Marrow MSC vial directly upon arrival or after storing in liquid N2 in a 37 °C water bath for a maximum of 90 sec. 
4.    Quickly transfer the thawed contents from the cell vial to a 50-mL conical tube containing 5 mL of StromaLine Bone-Marrow MSC Medium, pre-warmed at 37 °C. Centrifuge at 500xg for 5 min. Discard the supernatant and resuspend the cell pellet in 20 mL StromaLine Bone-Marrow MSC Medium. This step generates a cell suspension with a density of ~50,000 cells/mL, sufficient to seed an entire 96-well plate. We recommend this density for optimal results.
5.    Carefully peel off the sealing adhesive foil from the StromaLine Assay Microtiter Plate. A liquid meniscus may form on top of the wells due to the negative pressure applied by removing the foil, but it pops and disappears within seconds. Using a P100 pipet, insert the pipet tip in the well and descend along the side wall until you reach the plastic ring inside the well. Aspirate carefully the storage saline buffer. Do not touch or aspirate right over the hydrogel to prevent damaging it. Avoid aspirating the storage buffer using a vacuum pump as the suction force may damage the hydrogel. The storage buffer is a Tris-based buffer, and if some remains in the well, it will not affect negatively culture development. 
6.    Add 200 µL/well of the cell suspension prepared in step 4 to the StromaLine Assay Microtiter Plate. This will achieve a cell density of ~10,000 cells/well, which we recommend for optimal results. Maintain the culture in a 37 °C humidified incubator under a 5% CO2 atmosphere. Change the medium after 2-3 days (200 µL/well). We recommend aspirating the medium using a multichannel pipet and not a vacuum pump as the suction force may damage the hydrogel. The culture can be maintained for at least 12 days. However, we recommend proceeding with the sequential seeding of the StromaLine Bone-Marrow EC cells at day 5.

Establishment of the MSC-EC co-culture

7.    Thaw the StromaLine Bone-Marrow EC Medium (delivered frozen) at 37 °C for upon arrival or prior to use. Do not refreeze. Prepare 50-mL aliquots and store at 4 °C for up to 1 month. Avoid repeating cycles of warming as this results in reduced activity of the growth factors used to supplement the medium and suboptimal culture growth. 
8.    Thaw the frozen StromaLine Bone-Marrow EC vial that has been stored in liquid N2 in a 37 °C water bath for a maximum of 90 sec.
9.    Quickly transfer the thawed contents from the cell vial to a 50-mL conical tube containing 5 mL of StromaLine Bone-Marrow EC Medium, pre-warmed at 37 °C. Centrifuge at 500xg for 5 min. Discard the supernatant and resuspend the cell pellet in 20 mL StromaLine Bone-Marrow EC Medium. This step generates a cell suspension with a density of 50,000 cells/mL, sufficient to seed an entire 96-well plate. We recommend this density for optimal results.
10.    Take the StromaLine Assay Microtiter Plate with the growing MSC supporting culture from the 37 °C incubator just prior to EC seeding. Using a P100 pipet, insert the pipet tip in the well and descend along the side wall until you reach the plastic ring inside the well. Aspirate carefully the culture medium. Do not touch or aspirate right over the hydrogel to prevent damaging the growing culture. Avoid aspirating the storage buffer using a vacuum pump as the suction force may damage the hydrogel. If a small amount of the culture medium remains in the well, it will not affect negatively the subsequent co-culture development.
11.    Add 200 µL/well of the cell suspension prepared in step 3 to the StromaLine Assay Microtiter Plate with the growing MSC supporting culture. This will achieve a cell density of 10,000 cells/well, which we recommend for optimal results. Maintain the culture in a 37 °C humidified incubator under a 5% CO2 atmosphere. Change the medium every 2-3 days (200 µL/well). We recommend aspirating the medium using a multichannel pipet and not a vacuum pump as the suction force may damage the hydrogel. The culture can be maintained for at least 7 days. 

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More detailed protocols for handling the assay plate and for cell seeding can be found in the 3DProSeed General Usage Manual (available upon request).

Cell Characterization

The StromaLine Bone-Marrow Vascular Niche system relies on the sequential seeding of bone-marrow-derived MSC and EC on the StromaLine Assay Microtiter Plate to support the growth of an EC network mimicking the bone marrow vascular niche. The stable expression of EGFP in the StromaLine Bone-Marrow EC allows monitoring in real time the growth of the EC network on the supporting MSC culture. EC outgrowth from the initially formed cell clusters onto the spindle-like underlying MSC can be observed already 3 days after EC seeding, and a well-formed EC network is developed by day 7 (Figure 1). The EC network extends in 3D, covering at least a volume of 200 µm in depth (Figure 2). No such EC network can be formed in the absence of the supporting MSC culture. Seeding EC alone in the StromaLine Assay Microtiter Plate, in the absence or presence of MSC-conditioned medium, results in spherical cell clusters, which show no signs of outgrowth and eventually die. 
The StromaLine Bone-Marrow Vascular Niche co-culture system creates an extracellular microenvironment with features of the vascular basement membrane. In particular, secretion and deposition of fibronectin and laminin-5 is observed in close proximity to the EC network (Figure 3). Allowing the co-cultures to develop for longer periods of time (16 days) results in enhanced deposition of fibronectin but reduced amounts of laminin-5.