Basement Membrane Matrix Encapsulated Cell Aggregation for Investigating Murine Spleen Tissue Formation

The overall goal of this protocol is to aggregate and encase cells within a semi-solid matrix. Embedded cell aggregates can then be used downstream in three-dimensional in vitro culture, or as a vehicle for in vivo transplantation experiments. The main advantage of this method is that it uses simple methodology and equipment to achieve cell aggregation.

It can also be applied to a range of cell types and numbers. This technique has revealed a specific cell type that is required for the spleen to regenerate. It could uncover further regulators of spleen tissue regeneration, or serve as a platform for broader tissue engineering studies.

Begin by pre-chilling a 200 microliter pipette tip box inside a minus 20 degrees Celsius freezer. Then submerge Parafilm in 80%ethanol for 10 minutes, followed by a 10 minute wash in PBS. To prepare the cell solution, aliquot the desired cell number into a 14 milliliter conical tube.

Centrifuge the cells at 200g and 4 degrees Celsius for 5 minutes. Then carefully aspirate the supernatant, leaving approximately 20 microliters of volume behind. The cell pellet can then be resuspended in the remaining supernatant volume.

Using a pre-chilled pipette tip, aspirate 2 microliters of ice-cold Matrigel. Gently twist and eject to remove the pipette tip. Stretch a pre-sterilized strip of Parafilm and place it over the end of the pipette tip, taking care not to pierce the film.

Continue to wrap the Parafilm around the side to ensure the tip is sealed. Then layer the cell solution gently over the Matrigel. Leave the pipette tip on ice and prepare a 14 milliliter conical tube with a 1.2 milliliter cluster tube placed inside.

The pipette tip can then be inserted inside the nested tube configuration, and centrifuged at 400g and 4 degrees Celsius for 5 minutes. Incubate the tube at 37 degrees Celsius for 15 minutes. The tube can be placed on ice until further required.

Carefully remove the Parafilm from the pipette tip, then insert a thin wire plunger, and expel the Matrigel plug into tissue culture medium. The aggregated cells can be cultured at 37 degrees Celsius and 5%carbon dioxide. Shave the fur using electric clippers and swab the skin with 80%ethanol.

Make a two centimeter incision in the skin perpendicular to the spine, and expose the peritoneal wall. A second smaller incision is then made in the peritoneal wall above the kidney. Apply pressure and exteriorize the kidney.

Ensure the kidney is kept moist by regularly applying sterile PBS. Pinch the kidney capsule with a pair of ultra-fine forceps, and using a second pair, gently tear in opposing directions. Carefully separate the capsule membrane from the kidney parenchyma.

Lift the kidney capsule with one pair of forceps and slowly slide the pipette tip through the opening. Gently insert a wire plunger into the pipette tip and expel the Matrigel plug. Moisten the kidney with PBS and reinternalize into the peritoneal cavity.

Close the peritoneal wall with 5-O-Vicryl sutures, and close the skin incision using an AutoClip applier and two 9 millimeter wound clips. A key step in this protocol is aggregating cells within a semi-solid matrix. A middle layer positioning is achieved through optimal centrifugation speeds.

Higher speeds propel cells to the very tip of the pipette, while insufficient speeds prohibits cell movement through the matrix. Following solidification, the Matrigel construct can be ejected from the pipette tip. Spleen stromal constructs which are cultured in vitro form three-dimensional spherical organoid structures.

Organoids display a non-hollow structure with cells present across the entire diameter of the tissue. They are comprised of three broad cell populations, including white blood cells, red blood cells, and non-hemopoietic stromal and endothelial cells. Constructs can also be transplanted under the kidney capsule for tissue regeneration studies.

After four weeks, organized spleen tissue structure can be observed, including central arterioles, B cell follicles, fibroblastic reticular cells, red pulp myeloid cells, marginal zone metallophilic macrophages, red pulp sinusoids, and follicular dendritic cell networks, T-cell zone, red pulp macrophages, and marginal zone reticular cells. While attempting cell aggregation and encapsulation, it is important to remember to work quickly and to keep all materials on ice. The most important step for transplantations is expelling the Matrigel plug whilst withdrawing the pipette tip from the kidney.

Care must be taken not to perforate the capsule membrane or to rupture the kidney parenchyma. This protocol was used to investigate the requirements for two stromal cell populations in spleen tissue regeneration. In this representative experiment, only aggregates depleted of platelet-derived growth factor receptor-beta positive MAdCAM negative cells failed to initiate tissue growth, suggesting this particular cell population is essential for the spleen to regenerate.