Name: basement membrane matrix encapsulated cell aggregation for investigating murine spleen tissue formation
Uploaded: 2024-06-28T13:10:00
Description: 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 ...

This research was supported by the National Health and Medical Research Council of Australia (#GNT1078247).

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
<ol>
	<li>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 &#62;10 min. 
	NOTE: This protocol can be adapted to different mouse strains, ...

<ol>
	<li>Jarcho, S., Andersen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traumatic+autotransplantation+of+splenic+tissue.">Traumatic autotransplantation of splenic tissue.</a> American Journal of Pathology. 15 (5), 527-546 (1939).</li><li>Storsteen, K. A., ReMine, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rupture+of+the+spleen+with+splenic+implants:+splenosis.">Rupture of the spleen with splenic implants: splenosis.</a> Annals of Surgery. 137 (4), 551-557 (1953).</li><li>Mizrahi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posttraumatic+autotransplantation+of+spleen+tissue.">Posttraumatic autotransplantation of spleen tissue.</a> Archives of Surgery. 124 (7), 863 (1989).</li><li>Miko, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spleen+autotransplantation.+Morphological+and+functional+follow-up+after+spleen+autotransplantation+in+mice:+A+research+summary.">Spleen autotransplantation. Morphological and functional follow-up after spleen autotransplantation in mice: A research summary.</a> Microsurgery. 27 (4), 312-316 (2007).</li><li>Grikscheit, T. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+spleen+protects+against+overwhelming+Pneumococcal+sepsis+in+a+rodent+model.">Tissue-engineered spleen protects against overwhelming Pneumococcal sepsis in a rodent model.</a> Journal of Surgical Research. 149 (2), 214-218 (2008).</li><li>Pabst, R., Westermann, J., Rothkotter, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoarchitecture+of+regenerated+splenic+and+lymph+node+transplants.">Immunoarchitecture of regenerated splenic and lymph node transplants.</a> International Review of Cytology. 128, 215-260 (1991).</li><li>Tan, J. K. H., Watanabe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+spleen+tissue+regeneration+from+neonatal+spleen+capsule+requires+lymphotoxin+priming+of+stromal+cells.">Murine spleen tissue regeneration from neonatal spleen capsule requires lymphotoxin priming of stromal cells.</a> The Journal of Immunology. 193 (3), 1194-1203 (2014).</li><li>Tan, J. K. H., Watanabe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+cell+subsets+directing+neonatal+spleen+regeneration.">Stromal cell subsets directing neonatal spleen regeneration.</a> Scientific Reports. 7 (1), 40401 (2017).</li><li>Gee, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spleen+organoid+units+generate+functional+human+and+mouse+tissue-engineered+spleen+in+a+murine+model.">Spleen organoid units generate functional human and mouse tissue-engineered spleen in a murine model.</a> Tissue Engineering Part A. 26 (7-8), 411-418 (2020).</li><li>Ueno, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factor+Tlx1+marks+a+subset+of+lymphoid+tissue+organizer-like+mesenchymal+progenitor+cells+in+the+neonatal+spleen.">Transcription factor Tlx1 marks a subset of lymphoid tissue organizer-like mesenchymal progenitor cells in the neonatal spleen.</a> Scientific Reports. 9 (1), 20408 (2019).</li><li>. Online data repository: Matrigel encapsulated cell aggregation for investigating murine spleen tissue formation Available from: <a target="_blank" href="https://osf.io/ehrbw/?view_only=7d6a8e05c84144d3a12d36ffe7f94f01">https://osf.io/ehrbw/?view_only=7d6a8e05c84144d3a12d36ffe7f94f01</a> (2023)</li></ol>

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...

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 capacity1,2. 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 function4,5. 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 PDGFR&#946;+ 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 scaffold5,9. 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).

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			<td>96 Well Polypropylene 1.2 mL Cluster Tubes</td>
			<td>Corning</td>
			<td>CLS4401</td>
			<td>For placing inside a 14 ml conical tube</td>
		</tr>
		<tr>
			<td>B-mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td>Stock 55 mM, use at 50 uM</td>
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		<tr>
			<td>Collagenase D</td>
			<td>Roche</td>
			<td>11088858001</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Sigma-Aldrich</td>
			<td>C5138</td>
			<td>From Clostridium histolyticum</td>
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		<tr>
			<td>Deoxyribonuclease I (DNase I)</td>
			<td>Sigma-Aldrich</td>
			<td>D4513</td>
			<td>Deoxyribonuclease I from bovine pancreas,Type II-S, lyophilized powder, Protein &#8805;80 %, &#8805;2,000 units/mg protein</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)</td>
			<td>Sigma-Aldrich</td>
			<td>D5796</td>
			<td>4500 mg/L Glucose, L-Glutamine, and Sodium Bicarbonate, without Sodium Pyruvate, Liquid. Sterile Filtered.</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td>Without calcium chloride and magnesium chloride, sterile-filtered</td>
		</tr>
		<tr>
			<td>Eclipse 200 &#956;l Pipette Tips</td>
			<td>Labcon</td>
			<td>1030-260-000</td>
			<td>Bevel Point</td>
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		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>26140-079</td>
			<td>Lot# 1382243</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>Stock 100X, use at 1X</td>
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		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354263</td>
			<td>Matrigel matrix basement membrane High Concentration, Lot# 7330186</td>
		</tr>
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			<td>MEM Non-essential Amino Acids</td>
			<td>Gibco</td>
			<td>11140076</td>
			<td>Stock 100X, use at 1X</td>
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		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>Stock 10,000 units/ml Penicillin, 10,000 ug/ml Streptomycin</td>
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			<td>Reversible Cell Strainer</td>
			<td>STEMCELL Technologies</td>
			<td>27216</td>
			<td>70 &#956;m</td>
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			<td>Ring Tweezers</td>
			<td>NAPOX</td>
			<td>A-26</td>
			<td>Ring size: 3 mm</td>
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			<td>Rock Inhibitor (Y-27632)</td>
			<td>MedChemExpres</td>
			<td>HY-10071</td>
			<td></td>
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			<td>Thermofisher Heraeus Megafuge 40R Centrifuge</td>
			<td>Thermofisher</td>
			<td></td>
			<td>Acceleration and deceleration speeds were set to 8</td>
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		<tr>
			<td>Ultra Fine Tweezers</td>
			<td>EMS</td>
			<td>78340-51S</td>
			<td>Style 51S. Antimagnetic/anti-acid SA low carbon austenitic steel tweezers are corrosion resistant. Anti-glare satin finish.</td>
		</tr>
		<tr>
			<td>Vicryl 5/0 Suture Ligapak Reel</td>
			<td>Ethicon</td>
			<td>J283G</td>
			<td></td>
		</tr>
		<tr>
			<td>Wiretrol II Long Wire Plunger</td>
			<td>Drummond</td>
			<td>5-000-2002-L</td>
			<td>Stainless Steel Plunger, 25 &#38; 50 &#956;L/WRTL II, Long&#160;</td>
		</tr>
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			<td>Wound Clip Applier</td>
			<td>MikRon</td>
			<td>427630</td>
			<td></td>
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			<td>Wound Clips</td>
			<td>MikRon</td>
			<td>427631</td>
			<td>9 mm</td>
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basement membrane matrix encapsulated cell aggregation for investigating murine spleen tissue formation

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 PDGFR&#946;+MAdCAM-1- neonatal stromal cells are required for spleen tissue regeneration under animal transplantation models.

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...

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.

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

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 ...

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