Method Article

An Orthotopic Endometrial Cancer Model with Retroperitoneal Lymphadenopathy Made From In Vivo Propagated and Cultured VX2 Cells

DOI:

10.3791/59340

⸱

September 12th, 2019

 ,  ,  ,  ,  ,  ,  ,  ,  , 
1Institute of Medical Science, University of Toronto, 2Department of Obstetrics and Gynecology, University of Toronto, 3Guided Therapeutics Laboratory, TECHNA Institute, University Health Network, 4Department of Pathology, Princess Margaret Cancer Center, University Health Network, 5Department of Medical Biophysics, University of Toronto, 6Princess Margaret Cancer Center, University Health Network, 7Techna Institute, University Health Network, 8Department of Surgery, University of Toronto, 9Department of Otolaryngology, Head and Neck Surgery, University of Toronto, 10Division of Gynecologic Oncology, University of Toronto /Princess Margaret Cancer Center, University Health Network, 11Princess Margaret Cancer Center, University Health Network

In This Article

Summary

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This protocol presents a standardized method to grow VX2 cells in culture and to create an orthotopic VX2 model of endometrial cancer with retroperitoneal lymph node metastases in rabbits. Orthotopic endometrial cancer models are important for the pre-clinical study of novel imaging modalities for the diagnosis of lymph node metastases.

Abstract

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Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or without adjuvant therapy depending on lymph node involvement as determined by lymphadenectomy. Lymphadenectomy is a morbid procedure, which has not been shown to have a therapeutic benefit in many patients, and thus a new method to diagnose lymph node metastases is required. To test novel imaging agents, a reliable model of endometrial cancer with retroperitoneal lymph node metastases is needed. The VX2 endometrial cancer model has been described frequently in the literature; however, significant variation exists with respect to the method of model establishment. Furthermore, no studies have reported on the use of cultured VX2 cells to create this model as only cells propagated in vivo have been previously used. Herein, we present a standardized surgical method and post-operative monitoring method for the establishment of the VX2 endometrial cancer model and report on the first use of cultured VX2 cells to create this model.

Introduction

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Endometrial cancer, or cancer of the lining of the uterus, is the second most common gynecologic malignancy worldwide and the most common malignancy in developed nations1. The incidence of endometrial cancer has steadily increased, rising by 2.3% per year between 2005-2013 with a corresponding 2.2% increase in mortality1,2,3. The diagnosis of lymph node metastases is paramount as the presence of positive lymph nodes is a strong negative predictor of survival4,5,6,7 and can guide the administration of adjuvant therapy8,9,10,11,12,13. Lymph node metastases are currently diagnosed by surgically removing the lymphatic tissue overlying the major blood vessels in the pelvis and abdomen. This procedure, known as a lymphadenectomy, is controversial due to conflicting survival data from two large trials14,15,16,17,18 and the known risk of intra-operative15,19,20 and post-operative morbidity21,22,23. As current non-invasive imaging modalities do not have the required sensitivity and specificity to replace lymph node dissection24, there has been a push to develop new diagnostic imaging techniques. To test these novel techniques in a pre-clinical setting, a reliable model of endometrial cancer with retroperitoneal lymph node metastases is required.

The rabbit VX2 tumor model is a well-established model which has been used extensively to study multiple human solid organ tumors25 including lung26, head and neck27,28, liver29, kidney30, bone31,32, brain33, pancreas34 and uterus35,36,37. The VX2 model was originally developed in 1940 by Kidd and Rous38 by successfully transplanting a cottontail rabbit papilloma virus discovered by Shope in 193339. Since that time, the VX2 model has been maintained in vivo, requiring serial passage in the quadriceps muscle of White New Zealand rabbits40. More recently however, multiple groups have successfully grown VX2 cells in vitro40,41,42 and demonstrated the retained tumorigenecity of the cultured cell line31,42,43. VX2 tumors are histologically defined as anaplastic squamous cell carcinomas44 and contain glandular features which resemble adenocarcinoma26. Tumors are characterized by ease of implantation, rapid growth and hyper-vascularity44,45 and reliably metastasize, most commonly to regional and distant lymph nodes45. Similarities in uterine vascular and lymphatic anatomy46 as well as the orthotopic growth site ensure that the metastatic pattern of rabbit VX2 carcinoma mimics that of human endometrial cancer, making the VX2 model a reliable model for studying human metastatic disease. Furthermore, histologic features such as abnormal microvascular proliferation47 , as well as immunological48 and genetic similarities49,50 between humans and rabbits suggest that the tumor microenvironment may reflect that of human endometrial cancer.

Multiple groups have reported on the use of VX2 to create a model of endometrial cancer with retroperitoneal metastases with a high reported rate of success36,51,52; however, significant variation exists within the current literature with respect the method of model creation. Cell suspension doses as low as 4 x 105 cells/uterine horn51 and as high as 5 x 109 cells/uterine horn37,53 have been reported with no standard consensus on the required VX2 cellular dose. As well, a variety of inoculation methods have been reported including micro-surgical implantation of tumor into the uterine myometrium36, injection of VX2 cell suspension37,44,52,53 and in some cases, the addition of uterine horn suturing prior to innoculation52. Finally, no groups have reported the use of cultured VX2 cells to create this model. Thus, the purpose of this study is to demonstrate a successful standardized method of VX2 model creation and to report the first use of cultured VX2 cells to create a model of endometrial cancer with retroperitoneal metastases in a rabbit.

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Protocol

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Deep abdominal wall closure: Identify the apex of the peritoneal incision and grasp the peritoneum, rectus muscle and fascia with tissue forceps.All animal studies were conducted in Animal Resource Center (ARC) approved facilities of the University Health Network and in accordance with approved animal use and care protocols (AUP #3994/#4299). VX2 cell line was obtained from Dr. Aken’s Lab at the University Health Network.

1. Creation of in vitro VX2 cell line

  1. Harvest the VX2 tumor from rabbit quadricep muscle and growth in mouse flank.
    1. Thaw frozen VX2 tumor blocks (1 cm x 1 cm), mince on a culture plate using a scalpel blade and strain through a 70 µm filter by adding small amounts of Hanks Balanced Salt Solution (HBSS) sequentially to ensure all cells are strained for a final volume of 1 mL.
    2. Count cells and dilute with 0.9% phosphate buffered saline solution to a concentration of 1 x 107/mL and place on ice in a sterile tube.
      NOTE: Tumor blocks were obtained from a lab collaborator from previous propagation of VX2 tumors in rabbit quadriceps muscles.
    3. Anesthetize a female white New Zealand rabbit (weight 2.5-3.5 kg) with 2-5% inhaled isoflurane, remove the fur overlying the injection site and cleanse the injection site with povidone-iodine solution. Inject 500 µL of the prepared VX2 cell suspension (5 x 106 cells/mL) into the quadriceps muscle of the rabbit. Monitor the rabbit clinically for tumor growth starting on day 10.
    4. Euthanize the rabbits after tumors reach 1-1.5 cm in diameter (measured externally with calipers) using a veterinarian approved method. Confirm euthanasia by checking vital signs including heart rate and respiratory rate. Excise the tumor using sterile instruments after cleaning the area with betadine solution.
    5. In a biological safety cabinet using sterile instruments, mince the tumor in to small pieces (0.2 cm) on a 10 cm tissue culture dish using a scalpel blade. Pass the tumor fragments through a 70 µm cell strainer using 1 mL of HBSS as described in step 1.1.1. Count cells and dilute using 0.9% saline solution to obtain 7.5 x 105 cells in 200 µL.
    6. Anesthetize male NOD scid gamma mice (weight 25 g) using 4% isoflurane at 1 L/min and maintain anesthesia using 2% isoflurane. Once mice are anesthetized, inject 200 µL of the solution from step 1.1.5 into the subcutaneous tissue of the mouse flank using a 27 gauge needle.
  2. Harvesting and passaging of VX2 cells in vitro
    1. Monitor xenograft growth clinically twice weekly until tumors reach 1 cm in diameter using calipers.
    2. Euthanize mice by placing in a CO2 chamber or performing cervical dislocation after anesthetizing with 4% isoflurane gas. Excise tumors under sterile conditions in a biological safety cabinet.
    3. Mince tumors with a scalpel into 1-2 mm pieces in a 6 cm tissue culture dish containing 3 mL of Dulbecco’s Modified Eagle Medium (DMEM)/Ham’s F12+ 10% fetal bovine serum (FBS) media.
    4. Leave tumor fragments undisturbed in a 37 °C incubator with 5% CO2 for two days until cells begin to grow out from the tumor pieces. Subsequently, check cultured plates daily by light microscopy for cell confluency. Upon reaching 70% confluency, trypsinize cells by adding 1 mL of 0.05% trypsin to the flask and placing back in the 37 °C incubator for 5 min.
    5. Neutralize trypsin with 6 mL of DMEM/Ham’s F12 + 10% FBS medium, collect cells and centrifuge at 4 °C for 5 min at 300 x g. Remove supernatant and re-suspend pellet in 3.5 mL of new DMEM/Ham’s F12 + 10% FBS media. Seed fresh 6 cm collagen I coated plates with 1.6 x 106 cells per plate to achieve approximately 50% cell seeding density.
      ​NOTE: 50% cell density refers to the cells taking up 50% of the surface area of the plate when attached.
    6. Early passaging: Repeat the above process (trypsinizing, seeding) until cells have been passaged 5 times and then assess cell line purity with PCR using a quantitative PCR assay to distinguish rabbit genomic DNA from mouse genomic DNA (see step 1.3.1-1.3.4).
      NOTE: After 8 passages, cells can be propagated on non-collagen coated regular adherent tissue culture plates.
    7. Passage, trypsinize and freeze aliquots of cells in DMEM/HAM F12+30% FBS+10%DMSO at a concentration of 2 x 106 per vial. Store cells in liquid nitrogen until required for experiments.
  3. Confirmation of VX2 origin of surviving cells:
    1. Create a standard cure from purified genomic mouse and rabbit DNA (Step 1.3.2 and Step 1.3.3). Use primers which target species-specific sequences within the second open reading frame of the LINE-1 retrotransposon element.
      NOTE: Primer sequences for CRPV E6 are: 5’- GATCCTGGACCCAACCAGTGand 5’-CCTGCCGGTCCCTGATTTAT. The LINE-1 retrotransposon elements were used. Primer sequences for rabbit LINE-1 are: 5’-TCAGGAAACCCCAGAAAGTATGC and 5’-TTTGATTTCTTGAATGACCCAGTGT Primer sequences for mouse LINE-1 are: 5’-AATGGAAAGCCAACATTCACGTG and 5’-CCTTCCTTGACCAAGGTATCATTG
    2. To create a standard curve for CRPV E6, purify VX2 tumor cell lysate (Step 1.1.1) using a commercial kit following manufacturer’s protocol. Quantify this DNA using a commercial assay. Perform 6 serial dilutions from 225 pg/µL to 0.925 pg/µL in low EDTA-TE buffer. To create a standard curve for mouse genomic DNA, dilute 100 µg of commercial mouse genomic DNA in low EDTA-TE buffer in 5 dilutions from 125 pg/µL down to 0.0125 pg/µL.
    3. Place 4 µL from each diluted solution from the samples from step 1.3.2 in wells and run samples in a PCR thermocycler. Use the Cq values from each run along with the DNA amounts per well to calculate a standard curve using the thermocycler software auto-threshold settings.
    4. Use standard qPCR procedures to perform PCR with 40 cycles of 2-step cycling (98 °C and 60 °C) on a thermocycler. Establish threshold values, and set delta Ct values at >35 to ensure there is minimal mouse contamination in the rabbit VX2 cell line. The total volume of each reaction was 10 µL, consisting of 5 µL of 2x Mastermix, 1 µL of forward + reverse primers with final primer concentration in reaction at 250 nM, and 4 µL of DNA sample.
      NOTE: Please see references54,55 for details on performing PCR. Threshold values are established, and delta Ct values are set at >35 to ensure there is minimal mouse contamination in the rabbit VX2 cell line. The recommended level of VX2 DNA for reliable detection by qPCR is 1-10 pg.

2. VX2 cell culture and creation of cell suspension

  1. Thaw vials containing VX2 cells (frozen in step 1.2.7) in a water bath at 37 °C for 1 minute and transfer cells to a conical centrifuge tube with 10 mL of culture medium (1:1 DMEM/F-12 + 10% FBS and 1% Penicillin-streptomycin). Centrifuge for 8 min at 107 x g, discard the supernatant and re-suspend the cell pellet in 9 mL of media.
  2. Transfer re-suspended cells to a large culture flask. Incubate cells without shaking at 37 °C. Check cells daily for confluence using light microscopy and change culture media every three days.
  3. Creation of cultured VX2 cell suspension for injection
    1. Trypsinize cells by adding 3 mL of 0.25% trypsin to flask once cells have reached 80% confluence. Place flask in incubator (37 °C) for 5 min. Transfer solution to a conical centrifuge tube and centrifuge for 8 min at 107 x g and then remove supernatant.
    2. Wash the cell pellets 3 times with 9 mL of phosphate buffered saline, centrifuge and remove supernatant as above. Count the cells and dilute with 0.9% phosphate buffered saline solution to a concentration of 4 x 107 cells/mL and place in a sterile conical centrifuge tube on ice.
  4. Creation of in vivo propagated VX2 cell suspension for injection
    1. Thaw frozen VX2 tumor blocks (1 cm x 1 cm), mince on a culture plate using a scalpel blade and strain through a 70 µm filter as in step 1.1.1. Add small amounts of HBSS sequentially to ensure all cells are strained for a final volume of 1 mL. Count cells and dilute with 0.9% phosphate buffered saline to a concentration of 1 x107/mL and place on ice in a sterile tube.
      NOTE: Tumor blocks were obtained from a lab collaborator from previous propagation of VX2 tumors in rabbit quadriceps muscles.

3. Surgical Model Establishment

  1. Establishment of surgical anesthesia and pre-operative preparation
    1. Pre-medicate female white New Zealand rabbits (2.5-3.5 kg) 1 h prior to the planned surgical procedure using injections of acepromazine (1 mg/kg IM) and meloxicam (0.2 mg/kg SQ). Anesthetize a female white New Zealand rabbit (weight 2.5-3.5 kg) with 2-5% inhaled isoflurane, remove the fur overlying the injection site and cleanse the injection site with povidone-iodine solution.
    2. Intubate anesthetized rabbits using a laryngeal mask airway (LMA) and secure in place with tape, maintaining deep anesthesia by titrating the inhaled isoflurane dose between 2-5%. Monitor surgical anesthesia regularly throughout the procedure by checking vital signs (respiratory rate, capillary refill, oxygen saturation if available) and monitoring for signs suggestive of pain (movement, withdrawal from stimuli, noises) or light anesthesia (movement, chewing on LMA tube).
      NOTE: This should be done by someone with experience monitoring surgical anesthesia.
    3. Place a 22-gauge ear-vein catheter in the marginal dorsal vein. Administer cefazolin (20 mg/kg) intravenously 10 min prior to surgical skin incision.
    4. Clip hair over the pelvis and abdomen prior to placing the rabbit on the operating table, then place rabbits in a dorsal position on the operating table. Clean the surgical field using a 3-step surgical skin prep (betadine soap, chlorhexidine solution, betadine solution). Drape the surgical field with laparotomy drapes after land-marking the top of the pubic bone, leaving a 5 cm x 5 cm area of the lower abdomen exposed.
  2. Creation of the laparotomy incision and identification of the uterine horns
    1. Put on a surgical cap and face mask. Scrub hands using chlorhexidine or betadine surgical scrub solution. Using sterile technique, put on a sterile gown and sterile surgical gloves.
    2. Using a # 11-blade scalpel, make a 2.5 cm long incision 1 cm cranial to the symphysis pubis through skin and subcutaneous tissue of the rabbit abdomen. Incise the rectus fascia and dissect the rectus muscles laterally to expose the underlying peritoneum. Enter the peritoneum sharply after ensuring the undersurface is clear of bowel or other abdominal organs.
    3. Locate the uterine horns identifying the urinary bladder and sweeping a gloved finger superiorly, posteriorly and laterally over the apex of the bladder.
      NOTE: If necessary, the full bladder can be emptied using digital pressure. Once located, bring the uterine horns through the abdominal incision to rest on the abdominal wall.
    4. Using a 3-0 braided absorbable suture, perform a single suture ligation of each uterine horn approximately 1.5-2.0 cm distal to the cervices. Place the suture just medial to the uterine arteries, which run along the lateral aspect of each horn. Tie the sutures snugly to occlude the distal uterine horns (Figure 1).
  3. Myometrial VX2 inoculation
    1. Using a 27-gauge needle, inject 0.5 mL of the previously prepared VX2 cell suspension from either step 2.3.2 (for cultured VX2 model) or 2.4 (for in vivo propagated VX2 model) into the myometrium of each uterine horn proximal to the suture site (between the suture site and the cervix). Inject over 1 minute and ensure that cells are not being injected into the underlying uterine cavity. Anesthetize animals using inhaled isoflurane (2-5%) and an anesthetic machine with a Bain circuit.
    2. Apply pressure to the myometrial injection site for 30 s after injection to minimize leakage of cells. Inspect the injection and suture sites for hemostasis and place the uterine horns back into the abdomen.
  4. Closing the surgical incision
    1. Deep abdominal wall closure: Identify the apex of the peritoneal incision and grasp the peritoneum, rectus muscle and fascia with tissue forceps.
      1. To do this, anchor the suture at one apex of the incision by suturing superficial to deep on one side of the incision and deep to superficial on the other. Tie a knot. Using the attached suture, make running stitches perpendicular to the incision through the layers of the abdominal wall working step-wise along the incision from side to side. Tie the suture and cut.
    2. Superficial abdominal wall closure: Identify the apex of the skin incision and suture the abdominal skin using buried running subcuticular 3-0 absorbable poly-filament suture. Apply surgical glue to the closed incision to oppose the skin edges.
      1. To do this, anchor the suture at one apex of the incision, by suturing deep to superficial on one side of the incision and superficial to deep on the other. Tie a knot. Using the attached sutures, make running stitches in the dermal layer parallel to the incision working step-wise along the incision from side to side. Tie the suture and cut.
  5. Post-operative care
    1. Awakening from anesthesia: Turn off the isoflurane once the incision is closed and let the rabbit breath oxygen by mask while monitoring for signs of awakening (e.g., spontaneous movements, eye opening and chewing motions on the laryngeal mask airway). Extubate the rabbit once these signs are noted and provide  oxygen by mask and a warm blanket until the rabbits are alert and able to sit independently.
    2. Post-operative monitoring: Monitor rabbits twice a day for 4 days and daily for an additional 10 days post-operatively. To monitor, assess their general condition, their food and water intake, urine and fecal output, pain assessment, weight, vital signs (heart rate, respiration rate) and their surgical site looking for swelling, erythema, discharge or dehiscence.
    3. Post-operative pain control: Administer Meloxicam daily (0.2 mg/kg SQ) for 48 h post-operatively and then as needed based on pain assessment. Administer buprenorphine immediately post-operatively and every 12 h (0.01-0.05 mg/kg SQ) for 24 h and then as needed based on pain assessment
    4. Antibiotics (enrofloxacin 5mg/kg IM) may be administered daily for seven days post-operatively to prevent wound infection as recommended by your veterinarian. Administer subcutaneous fluids (15 mL of SQ) twice a day as needed for dehydration and soft foods or other dietary supplements are provided daily as needed for weight loss.

4. Tumor Growth Monitoring

  1. Clinical Monitoring: Monitor rabbits every 2 days for clinical signs of tumor growth starting on post-operative day 14. Clinical signs of tumor growth can include decreased oral intake, lethargy, abdominal tenderness, palpable abdominal mass and loss of cecotrophy.
  2. Imaging and invasive monitoring
    1. CT imaging: On post-operative day 21-28, pre-medicate, anesthetize, and intubate rabbits as previously described in 3.1.1-3.1.2. Position rabbits in a dorsal position in a pre-clinical CT scanner and obtain images of the pelvis and abdomen after administering 10 mL of intravenous iohexol contrast agent.
    2. Surgical monitoring: Transfer rabbits to the operating room after imaging while still under general anesthetic. Position, prep and drape rabbits as previously described in step 3.1.4 and perform a repeat laparotomy incision approximately 1.5 cm in length through the previous incision. Identify the and the uterine horns and carefully examine for tumor. Close the incision and provide post-operative care as previously described in step 3.5.2-3.5.4.
    3. Rabbit model use: If rabbits are found to have tumors at the time of surgical monitoring, use rabbit endometrial cancer models for planned experiments. Use rabbits created from in vivo propagated cells for additional experiments at 4-weeks post-inoculation and use rabbit models created from cultured VX2 cells at approximately 5-6 weeks post-inoculation to account for slower tumor growth. Euthanize the rabbits after tumors reach 1-1.5 cm in diameter (measured externally with calipers) using a veterinarian approved method. Confirm euthanasia by checking vital signs including heart rate and respiratory rate. Excise the tumor using sterile instruments after cleaning the area with betadine solution.

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Results

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Twenty-eight rabbits were used for the creation of the endometrial cancer model. Rabbits had an average weight of 2.83 kg (2.71-3.58 kg) at the time of experiment. Uterine tumors successfully grew in 21 rabbits for an overall model success rate of 75%. Prior to the inclusion of uterine suturing in the protocol, the success rate was 57% compared to 81% after uterine suturing was added. Uterine suturing was added to the protocol after the 7th rabbit in response to the initial low...

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Discussion

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Herein, we have reported a standardized surgical method for the establishment of a VX2 endometrial cancer model and reported on the first use of cultured VX2 cells to create this model. The tumor take rate of 75% is lower than the 100% percent rate previously reported in the literature35,37,53,56; however, thw 90% rate of pathologically confirmed lymph node metastases is consistent with previou...

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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This study was funded by the Terry Fox Research Institute (PPG#1075), the Canadian Institute of Health Research (Foundation Grant #154326), the Canadian Cancer Society Research Institute (704718), Natural Sciences and Engineering Research Council of Canada, Сanada Foundation for Innovation and Princess Margaret Cancer Foundation.

I would like to thank Dr. Marguerite Akens for providing the initial VX2 cells for the establishment of the initial VX2 model and the frozen VX2 tumor blocks. I would like to thank Marco DiGrappa for helping to perform initial VX2 cell culture experiments and Lili Ding for helping with VX2 cell culture.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
11-blade scalpel, Sterile, DisposibleAspen Surgical (VWR)80094-086
22-gague ear vein catheterCDMV14332
3-0 absorbable poly-filament suture (Polysorb)Covidien356718
3-0 braided absorbable suture (Polysorb)Covidien356718
70uM cell strainer, Individually wrapped, NylonFalcon352350
Acepromazine (Atravet)CDMV1047
Betadine soap (Poviodone iodine 7.5%)CDMV4363
Betadine solution (Poviodone iodine 10%)UHN Stores457955
BuprenorphineMcGill University
CefazolinUHN in-patient pharmacyNo Cat # Needed
Chlorhexidine solutionCDMV119872
Corning BioCoatCellware, Collagen Type I, 100mm dishesCorning354450brand not important
Corning BioCoatCellware, Collagen Type I, 24-well platesCorning354408brand not important
Corning BioCoatCellware, Collagen Type I, 6-well platesCorning354400brand not important
Corning Matrigel Basement Membrane Matrix, *LDEV-free, 10 mLCorning354234
DMEM/HAM F12 1:1Life Technologies11320brand not important
DMSOCaledon Lab Chem1/10/4100
Enrofloxacin (Baytril injectable)CDMV11242
Falcon TubeCorning Centri-Star430828
Fetal Bovine Serum, Qualified, Canadian Origin, 500mlLife Technologies12483020brand/source not important
IsofluraneUHN in-patient pharmacyNo Cat # Needed
Isohexol contrastGE Healthcare407141210
Meloxicam (Metacam 0.5%)CDMV104674
Normal SalineHouse Brand (UofT Medstore)1011
PBSMulticell or Sigma331-010-CL or D8537-500mL
Penicillin/Streptomycin (100mL; 10000U Penicillin, 10000ug Streptomycin)Corning-CellgroCA45000-652
Sterile Hanks Balanced Salt Solution (-Ca++, -Mg++, -Phenol Red)T.C.M.F (Dr Bristow)28-Jan-11
Surgical Glue (Tissue Adhesive)3M Vetbond14695B
Trypsin (0.25%), Proteomics GradeSigmaT-6567-5X20UG
Trypsin-EDTA, 0.05%, 100mlWisent Inc325-542-ELbrand not important

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VX2 Endometrial CancerIn Vivo Propagated CellsUterine Horn InjectionSurgical Model EstablishmentLymph Node MetastasisCell Culture PreparationHistological AnalysisSterile Surgical Technique

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