<meta charset="utf8"></head><body>인간 HSPC를 치사량이하로 조사된 마우스의 대퇴골에 주입

<ol>
	<li>Dancey, J. T., Deubelbeiss, K. A., Harker, L. A., Finch, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+kinetics+in+man.">Neutrophil kinetics in man.</a> J Clin Invest. 58 (3), 705-715 (1976).</li><li>Purton, L. E., Scadden, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limiting+factors+in+murine+hematopoietic+stem+cell+assays.">Limiting factors in murine hematopoietic stem cell assays.</a> Cell Stem Cell. 1 (3), 263-270 (2007).</li><li>Laurenti, E., G&#246;ttgens, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+haematopoietic+stem+cells+to+complex+differentiation+landscapes.">From haematopoietic stem cells to complex differentiation landscapes.</a> Nature. 553 (7689), 418-426 (2018).</li><li>Cosgrove, J., Hustin, L. S. P., de Boer, R. J., Peri&#233;, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoiesis+in+numbers.">Hematopoiesis in numbers.</a> Trends Immunol. 42 (12), 1100-1112 (2021).</li><li>Mazurier, F., Doedens, M., Gan, O. I., Dick, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+myeloerythroid+repopulation+after+intrafemoral+transplantation+of+NOD-SCID+mice+reveals+a+new+class+of+human+stem+cells.">Rapid myeloerythroid repopulation after intrafemoral transplantation of NOD-SCID mice reveals a new class of human stem cells.</a> Nat Med. 9 (7), 959-963 (2003).</li><li>McKenzie, J. L., Gan, O. I., Doedens, M., Dick, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+short-term+repopulating+stem+cells+are+efficiently+detected+following+intrafemoral+transplantation+into+NOD/SCID+recipients+depleted+of+CD122++cells.">Human short-term repopulating stem cells are efficiently detected following intrafemoral transplantation into NOD/SCID recipients depleted of CD122+ cells.</a> Blood. 106 (4), 1259-1261 (2005).</li><li>Notta, F., 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=Isolation+of+single+human+hematopoietic+stem+cells+capable+of+long-term+multilineage+engraftment.">Isolation of single human hematopoietic stem cells capable of long-term multilineage engraftment.</a> Science. 333 (6039), 218-221 (2011).</li><li>Belluschi, S., 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=Myelo-lymphoid+lineage+restriction+occurs+in+the+human+haematopoietic+stem+cell+compartment+before+lymphoid-primed+multipotent+progenitors.">Myelo-lymphoid lineage restriction occurs in the human haematopoietic stem cell compartment before lymphoid-primed multipotent progenitors.</a> Nat Commun. 9 (1), 4100 (2018).</li><li>McDermott, S. P., Eppert, K., Lechman, E. R., Doedens, M., Dick, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+human+cord+blood+engraftment+between+immunocompromised+mouse+strains.">Comparison of human cord blood engraftment between immunocompromised mouse strains.</a> Blood. 116 (2), 193-200 (2010).</li><li>Billerbeck, E., 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=Development+of+human+CD4+FoxP3++regulatory+T+cells+in+human+stem+cell+factor-,+granulocyte-macrophage+colony-stimulating+factor-,+and+interleukin-3-expressing+NOD-SCID+IL2R&#947;(null)+humanized+mice.">Development of human CD4+FoxP3+ regulatory T cells in human stem cell factor-, granulocyte-macrophage colony-stimulating factor-, and interleukin-3-expressing NOD-SCID IL2R&#947;(null) humanized mice.</a> Blood. 117 (11), 3076-3086 (2011).</li><li>Rongvaux, A., 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=Development+and+function+of+human+innate+immune+cells+in+a+humanized+mouse+model.">Development and function of human innate immune cells in a humanized mouse model.</a> Nat Biotechnol. 32 (4), 364-372 (2014).</li><li>McIntosh, B. E., 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=Nonirradiated+NOD,B6.SCID+Il2r&#947;&#8722;/&#8722;+KitW41/W41+(NBSGW)+Mice+support+multilineage+engraftment+of+human+hematopoietic+cells.">Nonirradiated NOD,B6.SCID Il2r&#947;&#8722;/&#8722; KitW41/W41 (NBSGW) Mice support multilineage engraftment of human hematopoietic cells.</a> Stem Cell Reports. 4 (2), 171-180 (2015).</li><li>Notta, F., Doulatov, S., Dick, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engraftment+of+human+hematopoietic+stem+cells+is+more+efficient+in+female+NOD/SCID/IL-2Rgc-null+recipients.">Engraftment of human hematopoietic stem cells is more efficient in female NOD/SCID/IL-2Rgc-null recipients.</a> Blood. 115 (18), 3704-3707 (2010).</li><li>Futrega, K., Lott, W. B., Doran, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+bone+marrow+HSC+transplantation+enhances+local+engraftment+at+the+expense+of+systemic+engraftment+in+NSG+mice.">Direct bone marrow HSC transplantation enhances local engraftment at the expense of systemic engraftment in NSG mice.</a> Sci Rep. 6, 23886 (2016).</li><li>Mende, N., 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=Unique+molecular+and+functional+features+of+extramedullary+hematopoietic+stem+and+progenitor+cell+reservoirs+in+humans.">Unique molecular and functional features of extramedullary hematopoietic stem and progenitor cell reservoirs in humans.</a> Blood. 139 (23), 3387-3401 (2022).</li><li>Medyouf, H., 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=Myelodysplastic+cells+in+patients+reprogram+mesenchymal+stromal+cells+to+establish+a+transplantable+stem+cell+niche+disease+unit.">Myelodysplastic cells in patients reprogram mesenchymal stromal cells to establish a transplantable stem cell niche disease unit.</a> Cell Stem Cell. 14 (6), 824-837 (2014).</li><li>Eppert, 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=Stem+cell+gene+expression+programs+influence+clinical+outcome+in+human+leukemia.">Stem cell gene expression programs influence clinical outcome in human leukemia.</a> Nat Med. 17 (9), 1086-1093 (2011).</li><li>Sanchez, P. V., 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=A+robust+xenotransplantation+model+for+acute+myeloid+leukemia.">A robust xenotransplantation model for acute myeloid leukemia.</a> Leukemia. 23 (11), 2109-2117 (2009).</li><li>Li, J., Chen, H., Zhao, S., Wen, D., Bi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+intrafemoral+orthotopic+xenografts+of+peripheral+blood+or+bone+marrow+from+acute+myeloid+and+acute+lymphoblastic+leukemia+patients:+clinical+characterization,+methodology,+and+validation.">Patient-derived intrafemoral orthotopic xenografts of peripheral blood or bone marrow from acute myeloid and acute lymphoblastic leukemia patients: clinical characterization, methodology, and validation.</a> Clin Exp Med. 23 (4), 1293-1306 (2023).</li><li>Dobson, S. M., 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=Relapse-fated+latent+diagnosis+subclones+in+acute+B+lineage+leukemia+are+drug+tolerant+and+possess+distinct+metabolic+programs.">Relapse-fated latent diagnosis subclones in acute B lineage leukemia are drug tolerant and possess distinct metabolic programs.</a> Cancer Discov. 10 (4), 568-587 (2020).</li><li>Marktel, S., 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=Intrabone+hematopoietic+stem+cell+gene+therapy+for+adult+and+pediatric+patients+affected+by+transfusion-dependent+&#223;-thalassemia.">Intrabone hematopoietic stem cell gene therapy for adult and pediatric patients affected by transfusion-dependent &#223;-thalassemia.</a> Nat Med. 25 (2), 234-241 (2019).</li></ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 mL Insulin Syringe with 29 G x 12.7 mm Needle</td>
			<td>BD</td>
			<td>324892</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL Insulin Syringe with 29 G x 0.5&#34; Needle</td>
			<td>BD</td>
			<td>324827</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL tube</td>
			<td>Fisherbrand</td>
			<td>509-GRD-PFB</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mL syringe&#160;</td>
			<td>HENKE SASS WOLF GMBH</td>
			<td>4020.000V0</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Round Bottom Polystyrene Test Tube with Cell Strainer Cap 12 x 75 mm</td>
			<td>Falcon</td>
			<td>352235</td>
			<td>FACS tube</td>
		</tr>
		<tr>
			<td>5 mL Round Bottom Polystyrene Test Tube with Snap Cap 12 x 75 mm</td>
			<td>Falcon</td>
			<td>352058</td>
			<td>FACS tube</td>
		</tr>
		<tr>
			<td>27 G 1/2&#34; needle&#160;</td>
			<td>BD&#160;</td>
			<td>300635</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m cell strainer for 50 mL tube</td>
			<td>Greiner Bio-one</td>
			<td>542040</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL tube</td>
			<td>Sarstedt Ltd</td>
			<td>62.547.254</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well round-bottom plate&#160;</td>
			<td>Falcon</td>
			<td>351177</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol Swab</td>
			<td>VITREX MEDICAL A/S</td>
			<td>520213</td>
			<td></td>
		</tr>
		<tr>
			<td>BD LSR Fortessa X-20 Cell Analyzer&#160;</td>
			<td>BD</td>
			<td></td>
			<td>flow cytometer</td>
		</tr>
		<tr>
			<td>Buphrenorphine</td>
			<td>Animalcare</td>
			<td>XVD190</td>
			<td></td>
		</tr>
		<tr>
			<td>CD14/PECy7 (Clone M5E2)</td>
			<td>biolegend</td>
			<td>301814</td>
			<td>Used at 1 in 1000</td>
		</tr>
		<tr>
			<td>CD19/Alexa 700 (Clone HIB19)</td>
			<td>biolegend</td>
			<td>302226</td>
			<td>Used at 1 in 300</td>
		</tr>
		<tr>
			<td>CD19/FITC (Clone HIB19)</td>
			<td>biolegend</td>
			<td>302206</td>
			<td>Used at 1 in 200</td>
		</tr>
		<tr>
			<td>CD3/APCCy7&#160;(Clone HIT3a)</td>
			<td>biolegend</td>
			<td>300318</td>
			<td>Used at 1 in 100</td>
		</tr>
		<tr>
			<td>CD33/APC (Clone P67.6)</td>
			<td>BD</td>
			<td>345800</td>
			<td>Used at 1 in 200</td>
		</tr>
		<tr>
			<td>CD45/BV510 (Clone HI30)</td>
			<td>biolegend</td>
			<td>304036</td>
			<td>Used at 1 in 500</td>
		</tr>
		<tr>
			<td>CD45/PECy5 (Clone 2D1)</td>
			<td>biolegend</td>
			<td>368526</td>
			<td>Used at 1 in 300</td>
		</tr>
		<tr>
			<td>CompBeads Anti-Mouse Ig, &#954;/Negative Control Compensation Particles Set</td>
			<td>BD</td>
			<td>552843</td>
			<td></td>
		</tr>
		<tr>
			<td>Dnase 1</td>
			<td>Worthington Biochemical</td>
			<td>LS002139</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>PAN-Biotech</td>
			<td>P40-37500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycophorin A (GlyA)/PE (Clone GA-R2)</td>
			<td>BD</td>
			<td>340947</td>
			<td>Used at 1 in 1000</td>
		</tr>
		<tr>
			<td>Iscove Modified Dulbecco Media (IMDM)</td>
			<td>PAN-Biotech</td>
			<td>P04-20250</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane (IsoFlo 100% w/w Inhalation Vapor, liquid)</td>
			<td>Zoetis</td>
			<td>115095</td>
			<td></td>
		</tr>
		<tr>
			<td>Microvette&#160;300 Lithium heparin LH, 300 &#181;L</td>
			<td>Sarstedt Ltd</td>
			<td>20.1309</td>
			<td>Mouse blood collection tube</td>
		</tr>
		<tr>
			<td>NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice&#160;</td>
			<td>Charles River&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pancoll human, Density: 1.077 g/mL</td>
			<td>PAN-Biotech</td>
			<td>P04-60500</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin&#160;</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>14190169</td>
			<td></td>
		</tr>
	</tbody>
</table>

intrafemoral injection of human hematopoietic stem and progenitor cells into immunocompromised mice 

Blood has one of the highest regeneration rates in the human body, producing 1 &#215; 1012 cells per day in the adult human bone marrow1. Hematopoietic stem cells (HSCs) guarantee blood production over the lifespan by the process of hematopoiesis and are defined by their capacity to produce all blood cell types (multipotentiality) while maintaining themselves (self-renewal). Historically, the gold standard for testing the function of an HSC has always relied on transplantation, testing the ability of a donor population to reconstitute all blood lineages of a mouse long-term (commonly defined as a minimum of 20 weeks)2. A large body of functional work spanning several decades has demonstrated that the HSC compartment is heterogeneous in both lineage output and long-term reconstitution. The toolkit to study hematopoiesis has expanded considerably over the years, with many new techniques, including in vitro single-cell functional assays, single-cell -omics approaches, and lineage tracing3. The latter have conclusively demonstrated that the contributions of HSC and multipotent progenitors largely differ in native hematopoiesis and under the stress imposed by transplantation. All these techniques complement transplantation assays, which remain important to assess the long-term repopulation capacity of HSCs. In the context of the study of human hematopoiesis, xenotransplantation provides the only method to experimentally assess self-renewal in a whole-organism setting.
Xenotransplantation of HSCs is commonly performed using intravenous injection of cells into immunocompromised mice. However, HSCs are rare4 and access to human samples containing HSCs is limited. In 2003, the group of John Dick adapted a protocol for bone marrow aspiration and intrafemorally injected non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mice with Lin&#8722;CD34+ umbilical cord blood (CB) cells5. To our knowledge, there has been no reported formal comparison of intravenous versus intrafemoral injections in long-term and serial transplantation outcomes. However, compared directly with intravenous injections, intrafemoral injections provide larger graft sizes with the same number of transplanted cells6, at least in the short term. In addition, engraftment can be detected with many fewer hematopoietic stem and progenitor cells (HSPCs) transplanted. This is thought to be because intrafemoral delivery bypasses the need for HSCs to home to the bone marrow, which in the xenograft context is limiting due to a lack of cross-species reactivity for a number of receptors and cytokines. Via the use of intrafemoral injections, Notta and colleagues were the first to transplant single human HSCs7, though extra considerations need to be taken, as described in their methods. Intrafemoral delivery of HSPCs also has limitations. The injection itself disrupts and destroys part of the bone marrow, and therefore is not indicated for studies of the crosstalk between HSCs and their bone marrow microenvironment. Additionally, the maximum number of cells is limited by the volume of that bone cavity and that may be too few for some applications. As with every technique, its application in a specific experiment needs to be weighed up based on the benefits/disadvantages and the question being asked. In the context of xenotransplantation, if the aim of the experiment is to test the engraftment of a low number of human HSPCs with no assessment of microenvironment, intrafemoral delivery is usually preferred over intravenous injection.

<meta charset="utf8"></head><body>저자 스포트라이트: 건강한 인간 조혈의 수명 역학 탐구

면역저하 마우스에 인간 조혈모세포와 전구세포의 대퇴골내 주입 

Our interest is studying healthy human hematopoiesis across a whole lifetime from the fetus to the elderly. For example, we study the liver in the fetus and the bone marrow and peripheral blood in the adult. We think it is important to study the healthy context to help us understand what goes wrong in disease.There are many single cell Onyx approaches that have advanced the field in recent years, but these work alongside rather than replace transplantation experiments that test the function of stem cells. By injecting blood stem and progenitor cells directly into the femur, we bypass the homing phase of intravenous injections. The cells are already in their natural environment, and therefore we often have much higher engraftment.We want to provide a quantitative view of how blood stem cells evolve over human life, how they are affected by events such as inflammation or infections, and finally, how mutations accumulate with age, how they reshape blood stem cell function, and how this predisposes us to age-related disease.

Read this Scientific Article Protocol about Author Spotlight: Exploring the Lifespan Dynamics of Healthy Human Hematopoiesis at JoVE.com

Hematopoietic stem cells (HSCs) are defined by their lifelong ability to produce all blood cell types. This is operationally tested by transplanting cell ...

intrafemoral-injection-human-hematopoietic-stem-progenitor-cells-into

Research

JoVE Journal

Biology

Intrafemoral Injection of Human Hematopoietic Stem and Progenitor Cells into Immunocompromised Mice 

692 조회수.  University of Cambridge. 우리의 관심은 태아에서 노인에 이르기까지 전 생애에 걸쳐 건강한 인간 조혈을 연구하는 것입니다. 예를 들어, 우리는 태아의 간을 연구하고 성인의 골수 및 말초 혈액을 연구합니다. 우리는 질병에서 무엇이 잘못되었는지 이해하는 데 도움이 되는 건강한 맥락을 연구하는 것이 중요하다고 생각합니다.최근 몇 년 동안 이 분야를 발전시킨 많?...

비디오: 저자 스포트라이트: 건강한 인간 조혈의 수명 역학 탐구

Hematopoietic stem cells (HSCs) are defined by their lifelong ability to produce all blood cell types. This is operationally tested by transplanting cell populations containing HSCs into syngeneic or immunocompromised mice. The size and multilineage composition of the graft is then measured over time, usually by flow cytometry. Classically, a population containing HSCs is injected into the circulation of the animal, after which the HSCs home to the bone marrow, where they lodge and begin blood production. Alternatively, HSCs and/or progenitor cells (HSPCs) can be placed directly in the bone marrow cavity. 
This paper describes a protocol for intrafemoral injection of human HSPCs into immunodeficient mice. In short, preconditioned mice are anesthetized, and a small hole is drilled through the knee into the femur using a needle. Using a smaller insulin needle, cells are then injected directly into the same conduit created by the first needle. This method of transplantation can be applied in varied experimental designs, using either mouse or human cells as donor cells. It has been most widely used for xenotransplantation, because in this context, it is thought to provide improved engraftment over intravenous injections, therefore improving statistical power and reducing the number of mice to be used.

Intrafemoral injections allow for the engraftment of a small number of hematopoietic stem and progenitor cells (HSPCs), by placing the cells directly in the bone marrow cavity. Here we describe an experimental protocol of intrafemoral injection of human HSPCs into immunodeficient mice.

연구

생물학

Intrafemoral Injection of Cord Blood CD34+ Cells into Mice for Enhanced Engraftment

To begin, remove the cryovial containing cord blood CD34+cells from the freezer. Thaw the cells in a water bath before transferring them to a 50 milliliter tube. While shaking the tube dropwise, add 10 times the cells volume of prewarmed IMDM containing 50%FBS and 0.1 milligram per milliliter DNase to the tube.Centrifuge the cell suspension at 500 G for five minutes. Resuspend the pellet in 20 to 40 microliters of PBS containing penicillin and streptomycin. Store the cells on ice until use.Prepare a three milliliter syringe with a 27-gauge half-inch needle. Prepare a 0.5 milliliter insulin syringe with a 29-gauge 12.7 millimeter needle containing cord blood CD34+cell suspension. Next, prepare a one milliliter insulin syringe with a 29-gauge 0.5-inch needle with 100 microliters of buprenorphine.Transfer the anesthetized mouse to a nose cone and perform a toe pinch to confirm its readiness for the injection. Lay the mouse on its back and flex its hind legs. To secure the hind legs, place the thumb of the nondominant hand on the foot, the middle finger on the hip, and the index finger on the outside of the femur.Gently pluck the hair from around the kneecap and wipe the area with an alcohol swab. Now using a three milliliter syringe with a 27-gauge half-inch needle, gently drill a hole through the skin towards the femur. Once the needle is in the bone, rotate it clockwise until the whole needle is in the bone.Remove the needle using an anti-clockwise rotation until halfway out. Wipe around the needle with an alcohol swab and continue to turn the needle anti-clockwise for complete removal. Next, insert the 0.5 milliliter insulin syringe containing cell suspension into the femoral shaft.Once the needle is in the bone, release the grip on the leg and gently inject 25 microliters of suspension into the femur. Finally, inject the buprenorphine subcutaneously and return the mouse to its home cage, allowing it to recover from anesthesia.