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, ages, and the number of animals.</li>
	<li>Prepare sterile surgical instruments.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Repeat step 1.6 for any remaining tissue.</li>
	<li>Leave the tube on ice for 1 min to let the tissue settle to the bottom of the tube.</li>
	<li>Carefully discard the supernatant (containing hematopoietic cells) by passing the solution through a reversible 70 &#181;m cell strainer.</li>
	<li>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&#39;s Modified Eagle Medium (sDMEM) containing 1 mg/mL Collagenase IV, 40 &#181;g/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.</li>
	<li>To prepare a single cell suspension, enzymatically digest pooled tissue (up to 20) for 10 min at 37 &#176;C with constant rotation.</li>
	<li>Add 4 mL of freshly prepared sDMEM containing 1 mg/mL Collagenase D, 40 &#181;g/mL DNase I, and 2% FBS directly to the tube containing stromal tissue. Incubate for a further 10 min at 37 &#176;C with constant rotation.</li>
	<li>Halt the digestion by adding 8 mL of ice-cold PBS. Collect cells by centrifuging at 200 x g for 5 min at 4 &#176;C.</li>
	<li>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 &#176;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.</li>
</ol>
2. Matrix encapsulation of cell aggregates
<ol>
	<li>Pre-chill sterile P200 pipette tips inside a -20 &#176;C freezer.</li>
	<li>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.</li>
	<li>Prepare 0.5 x 106-2.5 x 106 cells by centrifuging at 200 x g for 5 min at 4 &#176;C. Carefully aspirate the supernatant, leaving approximately 20 &#181;L 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.</li>
	<li>Aspirate 2 &#181;L 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.</li>
	<li>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.</li>
	<li>Resuspend the cell pellet in the remaining PBS and layer the solution gently over the basement membrane matrix. Keep the construct on ice.</li>
	<li>Prepare a centrifugation tube by placing a 1.2 mL cluster tube inside a 14 mL conical tube.</li>
	<li>Place the pipette tip inside the nested tube configuration and centrifuge at 400 x g and 4 &#176;C for 5 min.</li>
	<li>Position the 14 mL conical tube in a vertical orientation and incubate at 37 &#176;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.</li>
</ol>
3. Three-dimensional organoid culture
<ol>
	<li>Carefully remove the laboratory film from the pipette tip.</li>
	<li>Insert a thin stainless steel wire plunger through the larger opening of the pipette tip.</li>
	<li>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 &#181;M Rock Inhibitor, 10 U/mL Penicillin/Streptomycin, and 50 &#181;M &#946;-mercaptoethanol. 
	NOTE: The tissue culture vessel and corresponding media volume can be adjusted as required. 
	CAUTION: NEAA, Penicillin/Streptomycin, and &#946;-mercaptoethanol are hazardous substances and must be handled inside a biosafety cabinet with appropriate personal protective equipment.</li>
	<li>Culture organoids at 37 &#176;C, 5% CO2, and 95% humidity for 4-12 weeks.</li>
	<li>Replace half the medium every 5 days. 
	NOTE: If organoids begin attaching to the tissue culture plate, transfer to a fresh well.</li>
</ol>
4. Kidney capsule transplantation
<ol>
	<li>Prepare sterile surgical instruments.</li>
	<li>Perform a subcapsular kidney transplantation of the basement membrane matrix plug:
	<ol>
		<li>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.</li>
		<li>Lay the mouse in a right lateral position.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Prepare the basement membrane matrix plug for transplantation by removing laboratory film from the pipette tip.</li>
		<li>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.</li>
		<li>Insert a wire plunger into the pipette tip and expel the matrix plug whilst simultaneously withdrawing the pipette tip from the kidney.</li>
		<li>Moisten the kidney with PBS and re-internalize.</li>
		<li>Close the peritoneal wall with one 5-0 Vicryl suture and close the skin with two autoclips.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.</li>
	<li>Monitor post-surgical recovery for the first two weeks or as required by Institutional Guidelines.</li>
</ol>

<ol><li>Jarcho, S., Andersen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Traumatic+autotransplantation+of+splenic+tissue%5BTitle%5D)+AND+%22American+Journal+of+Pathology%22%5BJournal%5D)">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/pubmed?term=((Rupture+of+the+spleen+with+splenic+implants%3A+splenosis%5BTitle%5D)+AND+%22Annals+of+Surgery%22%5BJournal%5D)">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/pubmed?term=((Posttraumatic+autotransplantation+of+spleen+tissue%5BTitle%5D)+AND+%22Archives+of+Surgery%22%5BJournal%5D)">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/pubmed?term=((Spleen+autotransplantation.+Morphological+and+functional+follow-up+after+spleen+autotransplantation+in+mice%3A+A+research+summary%5BTitle%5D)+AND+%22Microsurgery%22%5BJournal%5D)">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/pubmed?term=((Tissue-engineered+spleen+protects+against+overwhelming+Pneumococcal+sepsis+in+a+rodent+model%5BTitle%5D)+AND+%22Journal+of+Surgical+Research%22%5BJournal%5D)">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/pubmed?term=((Immunoarchitecture+of+regenerated+splenic+and+lymph+node+transplants%5BTitle%5D)+AND+%22International+Review+of+Cytology%22%5BJournal%5D)">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/pubmed?term=((Murine+spleen+tissue+regeneration+from+neonatal+spleen+capsule+requires+lymphotoxin+priming+of+stromal+cells%5BTitle%5D)+AND+%22The+Journal+of+Immunology%22%5BJournal%5D)">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/pubmed?term=((Stromal+cell+subsets+directing+neonatal+spleen+regeneration%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">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/pubmed?term=((Spleen+organoid+units+generate+functional+human+and+mouse+tissue-engineered+spleen+in+a+murine+model%5BTitle%5D)+AND+%22Tissue+Engineering+Part+A%22%5BJournal%5D)">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/pubmed?term=((Transcription+factor+Tlx1+marks+a+subset+of+lymphoid+tissue+organizer-like+mesenchymal+progenitor+cells+in+the+neonatal+spleen%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">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>Tourle, K., Rucinski, A., Grainger, A., Limnios, I. J., Gonzalez Ruiz, M., Tan, J. K. H. 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 authors have nothing to disclose.

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).

<table><tbody><tr><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></tr><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></tr><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 &amp;ge;80 %, &amp;ge;2,000 units/mg protein</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;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&#039;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 &amp;mu;l Pipette Tips</td><td>Labcon</td><td>1030-260-000</td><td>Bevel Point</td></tr><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></tr><tr><td>Matrigel</td><td>Corning</td><td>354263</td><td>Matrigel matrix basement membrane High Concentration, Lot# 7330186</td></tr><tr><td>MEM Non-essential Amino Acids</td><td>Gibco</td><td>11140076</td><td>Stock 100X, use at 1X</td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140122</td><td>Stock 10,000 units/ml Penicillin, 10,000 ug/ml Streptomycin</td></tr><tr><td>Reversible Cell Strainer</td><td>STEMCELL Technologies</td><td>27216</td><td>70 &amp;mu;m</td></tr><tr><td>Ring Tweezers</td><td>NAPOX</td><td>A-26</td><td>Ring size: 3 mm</td></tr><tr><td>Rock Inhibitor (Y-27632)</td><td>MedChemExpres</td><td>HY-10071</td><td></td></tr><tr><td>Thermofisher Heraeus Megafuge 40R Centrifuge</td><td>Thermofisher</td><td></td><td>Acceleration and deceleration speeds were set to 8</td></tr><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 &amp;amp; 50 &amp;mu;L/WRTL II, Long&amp;nbsp;</td></tr><tr><td>Wound Clip Applier</td><td>MikRon</td><td>427630</td><td></td></tr><tr><td>Wound Clips</td><td>MikRon</td><td>427631</td><td>9 mm</td></tr></tbody></table>

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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Developmental Biology

1.4K Views.  Bond University. 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.

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

A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of acute leukemia. However, the nature of this disease has not been well documented in the literature. Here, we provide a technical protocol for characterizing C1498 cells in vitro and for determining the nature of the induced leukemia in vivo. The first part of this procedure is focused on determining the hematopoietic lineage and the stage of differentiation of cultured C1498 cells. To achieve this, multi-parametric flow cytometric staining is used to detect hematopoietic cell markers. Immunofluorescence microscopy, cytochemistry and a May-Gr&#252;nwald Giemsa staining are then performed to assess the expression of myeloperoxidase, the activity of esterases and cellular morphology, respectively. The second part of this protocol is dedicated to describing the leukemia disease that is induced in vivo. The latter can be achieved by determining the frequencies of leukemic and inherent cells in the blood, hematopoietic organs (e.g., bone marrow and spleen) and non-lymphoid tissues (e.g., the liver and lungs) using specific staining and flow cytometry analyses. The nature of the leukemia is then confirmed using May-Gr&#252;nwald Giemsa staining and staining for specific esterases in the bone marrow. Here, we present the results that were obtained using this protocol in age-matched C1498- and PBS-injected mice.

This manuscript provides a technical procedure that can be used to characterize C1498 cell cultures in vitro and the acute leukemia induced in mice after their injection. Phenotypic and functional analyses are performed using flow cytometry, immunofluorescence microscopy, cytochemistry and May-Gr&#252;nwald Giemsa staining.

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of ...

Cancer Research

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

Bioengineering

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...

Immunology and Infection

Construction, Characterization, and Regenerative Application of Self-Assembled Human Mesenchymal Stem Cell Aggregates

Mesenchymal stem cells (MSCs), characterized by their self-renewal ability and multilineage differentiation potential, can be derived from various sources and are emerging as promising candidates for regenerative medicine, especially for regeneration of the tooth, bone, cartilage, and skin. The self-assembled approach of MSC aggregation, which notably constructs cell clusters mimicking the developing mesenchymal condensation, allows high-density stem cell delivery along with preserved cell-cell interactions and extracellular matrix (ECM) as the microenvironment niche. This method has been shown to enable efficient cell engraftment and survival, thus promoting the optimized application of exogenous MSCs in tissue engineering and safeguarding clinical organ regeneration. This paper provides a detailed protocol for the construction and characterization of self-assembled aggregates based on umbilical cord mesenchymal stem cells (UCMSCs), as well as an example of the cranial bone regenerative application. The implementation of this procedure will help guide the establishment of an efficient MSC transplantation strategy for tissue engineering and regenerative medicine.

This study describes a method to construct aggregates based on the self-assembly of human mesenchymal stem cells and identifies the morphological and histological characteristics for the regenerative treatment of cranial bone defects.

Mesenchymal stem cells (MSCs), characterized by their self-renewal ability and multilineage differentiation potential, can be derived from various sources ...

Decellularized Rat Spleen Matrix Preparation for Liver Tissue Engineering

Fabrication of Decellularized Spleen Matrix Derived from Rats

Liver transplantation is the primary treatment for end-stage liver disease. However, the shortage and inadequate quality of donor organs necessitate the development of alternative therapies. Bioartificial livers (BALs) utilizing decellularized liver matrix (DLM) have emerged as promising solutions. However, sourcing suitable DLMs remains challenging. The use of a decellularized spleen matrix (DSM) has been explored as a foundation for BALs, offering a readily available alternative. In this study, rat spleens were harvested and decellularized using a combination of freeze-thaw cycles and perfusion with decellularization reagents. The protocol preserved the microstructures and components of the extracellular matrix (ECM) within the DSM. The complete decellularization process took approximately 11 h, resulting in an intact ECM within the DSM. Histological analysis confirmed the removal of cellular components while retaining the ECM's structure and composition. The presented protocol provides a comprehensive method for obtaining DSM, offering potential applications in liver tissue engineering and cell therapy. These findings contribute to the development of alternative approaches for the treatment of end-stage liver disease.

Author Spotlight: Utilization of Decellularized Spleen Matrix for Bioartificial Livers

The decellularized spleen matrix (DSM) holds promising applications in the field of liver tissue engineering. This protocol outlines the procedure for preparing rat DSM, which includes harvesting rat spleens, decellularizing them through perfusion, and evaluating the resulting DSM to confirm its characteristics.

Liver transplantation is the primary treatment for end-stage liver disease. However, the shortage and inadequate quality of donor organs necessitate the ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent ...

Medicine

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. Three-dimensional (3D) cell culture techniques are a recent innovation developed to address the shortcomings of adherent cell culture. While several techniques for generating tissue analogues in vitro have been developed, these methods are frequently complex, expensive to establish, require specialized equipment, and are generally limited by compatibility with only certain cell types. Here, we describe a rapid and flexible protocol for aggregating cells into multicellular 3D spheroids of consistent size that is compatible with growth of a variety of tumor and normal cell lines. We utilize varying concentrations of serum and methyl cellulose (MC) to promote anchorage-independent spheroid generation and prevent the formation of cell monolayers in a highly reproducible manner. Optimal conditions for individual cell lines can be achieved by adjusting MC or serum concentrations in the spheroid formation medium. The 3D spheroids generated can be collected for use in a wide range of applications, including cell signaling or gene expression studies, candidate drug screening, or in the study of cellular processes such as tumor cell invasion and migration. The protocol is also readily adapted to generate clonal spheroids from single cells, and can be adapted to assess anchorage-independent growth and anoikis-resistance. Overall, our protocol provides an easily modifiable method for generating and utilizing 3D cell spheroids in order to recapitulate the 3D microenvironment of tissues and model the in vivo growth of normal and tumor cells.

Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions.

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. ...

Biology

Myeloid Innate Signaling Pathway Regulation by MALT1 Paracaspase Activity

Besides its function in lymphoid cells, which has been addressed by numerous studies, the paracaspase MALT1 also plays an important role in innate cells downstream of pattern recognition receptors. Best studied are the Dectin-1 and Dectin-2 members of the C-type lectin-like receptor family that induce a SYK- and CARD9-dependent signaling cascade leading to NF-&#954;B activation, in a MALT1-dependent manner. By contrast, Toll-like receptors (TLR), such as TLR-4, propagate NF-&#954;B activation but signal via an MYD88/IRAK-dependent cascade. Nonetheless, whether MALT1 might contribute to TLR-4 signaling has remained unclear. Recent evidence with MLT-827, a potent and selective inhibitor of MALT1 paracaspase activity, indicates that TNF- production downstream of TLR-4 in human myeloid cells is independent of MALT1, as opposed to TNF- production downstream of Dectin-1, which is MALT1 dependent. Here, we addressed the selective involvement of MALT1 in pattern recognition sensing further, using a variety of human and mouse cellular preparations, and stimulation of Dectin-1, MINCLE or TLR-4 pathways. We also provided additional insights by exploring cytokines beyond TNF-, and by comparing MLT-827 to a SYK inhibitor (Cpd11) and to an IKK inhibitor (AFN700). Collectively, the data provided further evidence for the MALT1-dependency of C-type lectin-like receptor &#8212;signaling by contrast to TLR-signaling.

MALT1 regulates innate immunity but how this occurs remains ill-defined. We used the selective MALT1 paracaspase inhibitor MLT-827 to unravel the contribution of MALT1 to innate signaling downstream of Toll-like or C-type lectin-like receptors, demonstrating that MALT1 regulates the production of myeloid cytokines, and downstream of C-type lectin-like receptors, selectively.

Besides its function in lymphoid cells, which has been addressed by numerous studies, the paracaspase MALT1 also plays an important role in innate cells ...

In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy

Differentiation and maturation of megakaryocytes occur in close association with the cellular and extracellular components of the bone marrow. These processes are characterized by the gradual appearance of essential structures in the megakaryocyte cytoplasm such as a polyploid and polylobulated nucleus, an internal membrane network called demarcation membrane system (DMS) and the dense and alpha granules that will be found in circulating platelets. In this article, we describe a standardized protocol for the in situ ultrastructural study of murine megakaryocytes using transmission electron microscopy (TEM), allowing for the identification of key characteristics defining their maturation stage and cellular density in the bone marrow. Bone marrows are flushed, fixed, dehydrated in ethanol, embedded in plastic resin, and mounted for generating cross-sections. Semi-thin and thin sections are prepared for histological and TEM observations, respectively. This method can be used for any bone marrow cell, in any EM facility and has the advantage of using small sample sizes allowing for the combination of several imaging approaches on the same mouse.

In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy

Here, we present a protocol to analyze ultrastructure of the megakaryocytes in situ using transmission electron microscopy (TEM). Murine bone marrows are collected, fixed, embedded in epoxy resin and cut in ultrathin sections. After contrast staining, the bone marrow is observed under a TEM microscope at 120 kV.

Differentiation and maturation of megakaryocytes occur in close association with the cellular and extracellular components of the bone marrow. These ...

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

Summary

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Fabrication of Decellularized Spleen Matrix Derived from Rats

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production

Myeloid Innate Signaling Pathway Regulation by MALT1 Paracaspase Activity

In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy