Exploratory Study On The Immunogenicity Of Biological Materials With Immune Cells Migration Test

Background: Required source of autologous skin in the process of skin grafts for burnpatients is considerably limited, especially in severe burn patients, but allogeneic orxenogeneic skin graft induces host rejection response after transplantation.Tissue-engineered skin because of their wide variety of sources, and regenerativecharacteristics in burn treatment has good prospects for clinical application. It has becomeone focus of the wound repair research in recent years. However, how to overcome immunerejection of allogeneic organ transplantation is the major challenge. At present, the securityissues of tissue engineered medical products has attracted the attention of the food and drugadministration and foreign and domestic health management organizations, especially thebiological evaluation of products for human use. Therefore, objective evaluation of theimmunogenicity of their products is a significant demand. This study evaluates theimmunogenicity of the tissue engineered medical products through cell migration tests. Amethod was initially established to evaluate the immunogenicity of biological materials.This study might also provide experimental evidences for the immunogenicity of theevaluation of new tissue-engineered skin material.Objective:1. Through rabbit blood lymphocyte migration test, detecting the dose effectrelationship between lymphocyte and stimulant. The study will establish one method toevaluate the immunogenicity of biological materials.2. The study will establish one method to evaluate the immunogenicity of biologicalmaterials by detecting the chemotactic effect of BALB/c mouse spleen lymphocytes.3. Based on our previous results, Langerhans cell morphology by scanning electronmicroscopy and transmission electron microscopy and the phenotype of CD86and CD196of Langerhans cells are observed. The opitimal protocol to induce the Langerhans cells invitro from CD34+cells is screemed out to provide effector cells for the Langerhans cell migration test.Methods:1. Effect of different stimulants on rabbit lymphocyte migration test1.1Take rabbit blood, isolate lymphocytes by density gradient centrifugation, use PHAand ConA as lymphocyte stimulant separately. Add different concentrations of stimulatingagents to cell suspension, and let lymphocytes growth at37℃with5%CO₂incubator for72hours, then collect the cell supernatant.1.2Make cell supernatant as a source of chemokines, induce rabbit lymphocytemigration, measure cell migration rate with Transwell, and compare the migration effects ofthe stimulants. Understand the lymphocyte migration effect of stimulants.2. Effect of different stimulants on mouse lymphocyte migration test2.1Take lymphocytes of BALB/c mouse spleen by grinding method, use PHA, ConAand CD3ε monoclonal antibody as lymphocytes stimulant separately. Add differentconcentrations of stimulating agents to cell suspension, and let lymphocytes growth at37℃with5%CO₂incubator for72hours, then collect the cell supernatant.2.2Make cell supernatant as a source of chemokines, induce mouse lymphocytemigration, measure cell migration rate with Transwell, and compare the migration effects ofthe stimulants. Understand the lymphocyte migration effect of stimulant.3. Effect of different biological material extracts on mouse lymphocyte migration test3.1Preparation of biological material extracts:（1） Soluble materials, the cattle Icollagen was selected and dissolved in a purity of>99.5%acetic acid solution, a smallamount of packaging, was stored at4°C.（2） Insoluble material, the acellular freeze-driedcow dermis was selected and immersed in RPMI-1640and incubated for24hours. Thecollected extract, was stored at-20℃.3.2Lymphocytes of BALB/c mouse spleen were separated by grinding method andcultured with different concentrations of biological material extracts. After72hours ofculture at37℃with5%CO₂incubator, the cell supernatant was collected.3.3The cell supernatant was used as a source of chemokines to induce mouselymphocyte migration in Transwell. The migration effects of the biological material extractswere evaluated to understand the lymphocyte migration effect of biological materials.4. Chemokine levels in the supernatant of mouse lymphocytes 4.1Lymphocytes of BALB/c mouse spleen were seperated by grinding method. PHA,ConA and CD3ε monoclonal antibody were used as lymphocytes stimulant. After72hoursof culture at37℃with5%CO₂incubator, the cell supernatant was collected.4.2ELISA: The OD450values of cell supernatant were recorded by microplate reader,the concentrations of different chemokines were calculated to analysis their effect on cellmigration.5. Langerhans cells identification5.1The mononuclear cells （MNCs） were separated from human umbilical cord bloodby using human lymphocyte separation medium. The CD34+cells were then furtherpurified from MNCs by direct CD34immunomagnetic beads and MACS.5.2With five protocols of different combinations of cytokines, the Langerhans cellswere induced from CD34+cells in vitro. Their morphological features were observed byscanning electron microscopy and transmission electron microscopy.5.3Combined with the Langerhans cells phenotypic identification of the data, detectthe phenotypes of CD196and CD86.Results:1. In rabbit lymphocytes test, when the concentration of PHA was5μg/ml, and theconcentration of PHA were5μg/ml and10μg/ml, the lymphocyte migration rates of bothagent groups were significantly higher than control group.2. In mouse lymphocytes test, there was no difference of cell migration rate betweenPHA groups and control group. When the concentration of ConA was20μg/ml, and theconcentration of CD3ε were2.5μg/ml and10μg/ml, the lymphocyte migration rates ofboth agent groups were significantly higher than control group.3. The cattle I collagen in the concentrations of5,10,100,200, and300μg/ml, had asignificantly higher lymphocyte migration rate compared with control group. Acellularbovine dermal extracts had no effect on lymphocytes migration test.4. There were obvious differences about chemokines levels in general. The levels ofXCL1, CCL4, CCL5and CCL3were significantly higher than control group, they playedimportant roles in cell migration. And there was no difference between the levels of IL-16,IL-8and CCL20and control group.5. By scanning electron microscopy and transmission electron microscopy results, combined with the phenotypic identification of existing data shows that the protocol â…£ isoptimal.Conclusions:1. In rabbit lymphocyte migration test, PHA and ConA could induce lymphocytemigration, the mobility were significantly higher than control group.2. There was no enhancement of PHA on mouse lymphocytes migration. However,PHA and CD3ε could induce lymphocyte migration, the mobility were significantly higherthan control group.3. PHA, ConA and CD3ε could induce mouse lymphocyte to secret various levels ofchemokines.4. The cattle I collagen could enhance lymphocyte migration.Acellular bovine dermalextracts had no enhancement in lymphocyte migrantion.5. By scanning electron microscopy and transmission electron microscopy results,combined with the phenotypic identification of existing data shows that the protocol â…£ isoptimal.