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

Summary

This article describes a protocol for aggregating and encapsulating spleen cells within a semi-solid basement membrane matrix. Basement membrane matrix constructs can be used in three-dimensional culture for studying organoid development, or for in vivo transplantation and tissue regeneration studies.

Abstract

The spleen is an immune organ that plays a key role in blood-borne immune responses. The anatomical or functional loss of this tissue increases susceptibility to severe blood infections and sepsis. Auto-transplantation of spleen slices has been used clinically to replace lost tissue and restore immune function. However, the mechanism driving robust and immunologically functional spleen tissue regeneration has not been fully elucidated. Here, we aim to develop a method for aggregating and encapsulating spleen cells within a semi-solid matrix in order to investigate the cellular requirements for spleen tissue formation. Basement membrane matrix encapsulated cell constructs are amenable to both in vitro tissue culture of three-dimensional organoids as well as transplantation under the kidney capsule to directly assess in vivo tissue formation. By manipulating the input cells for aggregation and encapsulation, we demonstrate that graft-derived PDGFRβ⁺MAdCAM-1^- neonatal stromal cells are required for spleen tissue regeneration under animal transplantation models.

Introduction

Traumatic rupture of the spleen and the appearance of multiple splenic nodules in the body was one of the first indications that spleen tissue harbored regenerative capacity^1,2. Spleen auto-transplantations were later introduced into the clinic to preserve spleen tissue in patients requiring emergency splenectomy³. Yet, despite being a part of clinical practice for decades, very little is known about how the spleen regenerates. Animal transplantation models have provided insight into multiple parameters of spleen regeneration and immune function^4,5. In particular, experimental modifications to the graft preparation method have allowed tissue regeneration to be studied in greater detail at the cellular and molecular level.

Transplantations involving whole spleen slices undergo a phase of mass necrosis before a new spleen structure is rebuilt⁶. The initial phase of graft necrosis suggests that the bulk of transplanted tissue largely consists of red and white blood cells and is unnecessary for spleen regeneration. This was investigated experimentally by excluding hematopoietic cells from spleen grafts before transplant under the mouse kidney capsule. Here, the non-leukocyte/non-erythrocyte fraction of the spleen, which includes stromal and endothelial cells, was shown to be sufficient to induce de novo tissue formation⁷. Spleen stromal tissue could be further processed into a single-cell suspension, enabling the use of cell sorting technologies to manipulate cellular graft composition. By selectively removing candidate cell types, two CD45^-TER-119^- stromal cell populations were identified that were indispensable for graft development: an endothelial-like CD31⁺CD105⁺MAdCAM-1⁺ cell population and a more broadly defined PDGFRβ⁺ mesenchymal cell population⁸.

The construction of grafts from spleen cells varies in terms of support materials and cell-loading processes. Tissue-engineered spleens have previously been prepared by loading splenic units onto a polyglycolic acid/poly-L lactic acid polymer scaffold^5,9. Interestingly, spleen stromal cells absorbed into a collagen sponge failed to engraft, whereas stromal cells aggregated and loaded over a collagen sheet facilitated spleen regeneration⁸. The resuspension of spleen stromal cells inside a Matrigel matrix has also been demonstrated to induce cell aggregation under three-dimensional culture conditions¹⁰. However, this method has not been tested for use in transplantation models. The overall goal of the current protocol is to forcibly aggregate and encapsulate spleen stromal cells directly within the basement membrane matrix, which subsequently can be transferred to a three-dimensional in vitro tissue culture system or used as a vehicle for animal model transplantations (Supplementary Figure 1).

Protocol

All animal procedures were conducted according to experimental protocols approved by the University of Queensland Animal Ethics Committee (UQBR/079/19).

1. Tissue collection and stromal cell preparation

1. Euthanize 0.5-1.5 day-old male and/or female BALB/c neonatal donor mice by induction of hypothermia by wrapping animals inside the tissue paper and placing them under crushed ice for >10 min.
   NOTE: This protocol can be adapted to different mouse strains, ages, and the number of animals.
2. Prepare sterile surgical instruments.
3. Lay the mouse in a right lateral position. Swab the skin with 80% ethanol and make a 1 cm incision with Iris surgical scissors to expose the peritoneal wall.
4. Make a second 0.5 cm incision above the spleen and use a pair of fine forceps to gently lift the tissue up. Use a pair of surgical scissors to cut the blood vessels and excise the tissue. Transfer the tissue into a Petri dish containing ice-cold Phosphate Buffered Saline (PBS). Remove any remaining connective tissue attached to the spleen.
   NOTE: A stereomicroscope can assist fine motor movements required for manipulating surgical instruments.
5. To dissociate whole tissues, transfer neonatal spleens (pools of 10 or less) into the inner rim of an inverted 14 mL conical tube cap. Mechanically disrupt tissues with a pressing motion using the plastic back end of a 1 mL syringe plunger.
6. Place and secure the cap tightly over a 14 mL conical tube containing 10 mL of cold PBS. Invert the tube 5x to wash all disrupted tissue from the cap into the tube.
7. Repeat step 1.6 for any remaining tissue.
8. Leave the tube on ice for 1 min to let the tissue settle to the bottom of the tube.
9. Carefully discard the supernatant (containing hematopoietic cells) by passing the solution through a reversible 70 µm cell strainer.
10. Recover the non-soluble stromal fraction by reversing the strainer and washing stromal tissue back into the 14 mL conical tube using freshly prepared 2 mL of supplemented Dulbecco's Modified Eagle Medium (sDMEM) containing 1 mg/mL Collagenase IV, 40 µg/mL DNase I and 2% Fetal Bovine Serum (FBS).
    CAUTION: Collagenase IV and Collagenase D are hazardous substances and must be handled inside a biosafety cabinet with appropriate personal protective equipment.
11. To prepare a single cell suspension, enzymatically digest pooled tissue (up to 20) for 10 min at 37 °C with constant rotation.
12. Add 4 mL of freshly prepared sDMEM containing 1 mg/mL Collagenase D, 40 µg/mL DNase I, and 2% FBS directly to the tube containing stromal tissue. Incubate for a further 10 min at 37 °C with constant rotation.
13. Halt the digestion by adding 8 mL of ice-cold PBS. Collect cells by centrifuging at 200 x g for 5 min at 4 °C.
14. Discard the supernatant, and wash cells twice by resuspending in 1 mL of cold PBS and centrifuging at 200 x g for 5 min at 4 °C. Keep cells on ice.
    NOTE: Cells can be counted and adjusted to a desired concentration. This may require optimization for different cell populations. Using this protocol, 0.5 x 10⁶- 1 x 10⁶ stromal cells/spleen are typically recovered, depending on the age of donor mice. Cells can optionally be stained and FACS sorted at this stage to define cell composition.

2. Matrix encapsulation of cell aggregates

1. Pre-chill sterile P200 pipette tips inside a -20 °C freezer.
2. Cut flexible laboratory film (e.g., Parafilm) into 1 cm x 2 cm pieces and sterilize by submerging in 80% ethanol for 10 min, followed by PBS for 10 min. Laboratory film can be prepared in advance and stored sterile.
3. Prepare 0.5 x 10⁶-2.5 x 10⁶ cells by centrifuging at 200 x g for 5 min at 4 °C. Carefully aspirate the supernatant, leaving approximately 20 µL of the remaining PBS volume. Keep the cell pellet on ice.
   NOTE: The remaining PBS can be gently aspirated without disturbing the cell pellet to confirm the volume.
4. Aspirate 2 µL of ice-cold basement membrane matrix into a pre-chilled P200 pipette tip, using a P20 pipettor.
   NOTE: A highly concentrated basement membrane matrix (e.g., Corning Matrigel Matrix High Concentration) assists in forming a strong solidified plug.
5. Gently twist to eject the pipette tip. Stretch a pre-sterilized strip of the laboratory film and place it over the end of the pipette tip, taking care not to pierce the film. Continue to wrap the tip in the film to seal the pipette tip. Carefully place the sealed tip directly on ice, taking care not to rupture the film.
6. Resuspend the cell pellet in the remaining PBS and layer the solution gently over the basement membrane matrix. Keep the construct on ice.
7. Prepare a centrifugation tube by placing a 1.2 mL cluster tube inside a 14 mL conical tube.
8. Place the pipette tip inside the nested tube configuration and centrifuge at 400 x g and 4 °C for 5 min.
9. Position the 14 mL conical tube in a vertical orientation and incubate at 37 °C for 15 min to allow the basement membrane matrix to solidify inside the pipette tip.
   NOTE: The 14 mL conical tube can be placed on ice until required for downstream application.

3. Three-dimensional organoid culture

1. Carefully remove the laboratory film from the pipette tip.
2. Insert a thin stainless steel wire plunger through the larger opening of the pipette tip.
3. Expel the matrix plug until released through the tip into one well of a non-treated 6-well tissue culture plate containing 2 mL of sDMEM supplemented with 10% FBS, 1x Glutamax, 1x Non-essential Amino Acids (NEAA), 10 µM Rock Inhibitor, 10 U/mL Penicillin/Streptomycin, and 50 µM β-mercaptoethanol.
   NOTE: The tissue culture vessel and corresponding media volume can be adjusted as required.
   CAUTION: NEAA, Penicillin/Streptomycin, and β-mercaptoethanol are hazardous substances and must be handled inside a biosafety cabinet with appropriate personal protective equipment.
4. Culture organoids at 37 °C, 5% CO₂, and 95% humidity for 4-12 weeks.
5. Replace half the medium every 5 days.
   NOTE: If organoids begin attaching to the tissue culture plate, transfer to a fresh well.

4. Kidney capsule transplantation

1. Prepare sterile surgical instruments.
2. Perform a subcapsular kidney transplantation of the basement membrane matrix plug:
   1. Anesthetize 8-week old BALB/c female recipient mouse with isoflurane following Institutional Animal Ethics Guidelines.
      CAUTION: Isoflurane is a hazardous substance. Waste gas must be scavenged to prevent entry into the workspace environment.
   2. Lay the mouse in a right lateral position.
   3. Shave hair from the surgery site and disinfect the skin following Institutional Guidelines.
      NOTE: It is recommended that the surgical site be disinfected three times in a circular motion with alternating iodine-based or chlorhexidine-based and alcohol-based scrub.
   4. Make a 2 cm incision in the skin perpendicular to the spine to expose the peritoneal wall.
      NOTE: Fine surgical scissors or a scalpel blade can be used for skin incision. Users should follow Institutional Animal Ethics Guidelines or Standard Operating Procedures.
   5. Make a smaller 0.5 cm incision in the peritoneal wall above the kidney.
      NOTE: The incision should approximate the kidney width to ensure that the kidney remains exteriorized during transplantation.
   6. Exteriorize the kidney through the peritoneal opening by applying downward pressure using the thumb and index finger. A pair of ring tweezers can be used to assist exteriorization. Ensure the kidney is moist throughout the procedure by regularly applying sterile PBS using a cotton swab.
   7. Under a stereomicroscope, pinch the perirenal fat of the kidney capsule with a pair of bent ultra-fine forceps at one pole of the kidney. Use a second pair of bent ultra-fine forceps to gently tear the kidney capsule membrane upwards in an opposing direction, creating a small opening.
   8. Carefully insert the prong of one forceps under the capsule membrane. Use a slow sweeping motion to separate the capsule membrane from the kidney parenchyma.
   9. Prepare the basement membrane matrix plug for transplantation by removing laboratory film from the pipette tip.
   10. Lift the kidney capsule using one prong of the forceps and insert the pipette tip through the opening, pushing it towards the opposing pole of the kidney.
   11. Insert a wire plunger into the pipette tip and expel the matrix plug whilst simultaneously withdrawing the pipette tip from the kidney.
   12. Moisten the kidney with PBS and re-internalize.
   13. Close the peritoneal wall with one 5-0 Vicryl suture and close the skin with two autoclips.
   14. Administer analgesic (Buprenorphine at 0.05-0.1 mg/kg subcutaneously or following Institutional Animal Ethics Guidelines).
       CAUTION: Buprenorphine is a hazardous substance and must be handled with appropriate personal protective equipment.
3. Turn off the flow of isoflurane but keep the oxygen flow on to allow the mouse to breathe pure oxygen until it starts gaining consciousness.
4. Return the mouse to its cage. Keep the animal warm during recovery by partially placing the cage on top of a heating pad or under a heat lamp, and monitor until the mouse fully recovers from anesthesia.
5. Monitor post-surgical recovery for the first two weeks or as required by Institutional Guidelines.

Results

Cell aggregation is important for promoting cell-to-cell contact and signaling. Encasing cell aggregates inside the basement membrane matrix supported both 3-dimensional cultures for in vitro tissue organoid formation and facilitated the mechanical delivery of cells into the kidney capsule for graft transplantation. To establish these constructs, the basement membrane matrix was first maintained in a fluidic state under ice-cold conditions. Cell aggregation was subsequently achieved by layering a concentrated ce...

Discussion

The aggregation of neonatal spleen cells inside a semi-solid medium represents a viable method for generating spleen constructs. Similar basement membrane matrix-based protocols have been used to initiate three-dimensional spleen cultures¹⁰. Here, we demonstrate that spleen constructs are equally amenable to in vitro organoid culture systems as well as to in vivo transplantation models. Of note, the transplantation of in vitro cultured spleen organoids has not yet been t...

Materials

Name	Company	Catalog Number	Comments
96 Well Polypropylene 1.2 mL Cluster Tubes	Corning	CLS4401	For placing inside a 14 ml conical tube
B-mercaptoethanol	Gibco	21985023	Stock 55 mM, use at 50 uM
Collagenase D	Roche	11088858001
Collagenase IV	Sigma-Aldrich	C5138	From Clostridium histolyticum
Deoxyribonuclease I (DNase I)	Sigma-Aldrich	D4513	Deoxyribonuclease I from bovine pancreas,Type II-S, lyophilized powder, Protein ≥80 %, ≥2,000 units/mg protein
Dulbecco’s Modified Eagle’s Medium (DMEM)	Sigma-Aldrich	D5796	4500 mg/L Glucose, L-Glutamine, and Sodium Bicarbonate, without Sodium Pyruvate, Liquid. Sterile Filtered.
Dulbecco's Phosphate Buffered Saline (PBS)	Sigma-Aldrich	D8537	Without calcium chloride and magnesium chloride, sterile-filtered
Eclipse 200 μl Pipette Tips	Labcon	1030-260-000	Bevel Point
Fetal Bovine Serum (FBS)	Gibco	26140-079	Lot# 1382243
GlutaMAX	Gibco	35050061	Stock 100X, use at 1X
Matrigel	Corning	354263	Matrigel matrix basement membrane High Concentration, Lot# 7330186
MEM Non-essential Amino Acids	Gibco	11140076	Stock 100X, use at 1X
Penicillin/Streptomycin	Gibco	15140122	Stock 10,000 units/ml Penicillin, 10,000 ug/ml Streptomycin
Reversible Cell Strainer	STEMCELL Technologies	27216	70 μm
Ring Tweezers	NAPOX	A-26	Ring size: 3 mm
Rock Inhibitor (Y-27632)	MedChemExpres	HY-10071
Thermofisher Heraeus Megafuge 40R Centrifuge	Thermofisher		Acceleration and deceleration speeds were set to 8
Ultra Fine Tweezers	EMS	78340-51S	Style 51S. Antimagnetic/anti-acid SA low carbon austenitic steel tweezers are corrosion resistant. Anti-glare satin finish.
Vicryl 5/0 Suture Ligapak Reel	Ethicon	J283G
Wiretrol II Long Wire Plunger	Drummond	5-000-2002-L	Stainless Steel Plunger, 25 & 50 μL/WRTL II, Long
Wound Clip Applier	MikRon	427630
Wound Clips	MikRon	427631	9 mm