[생명공학] CD34+ HSC isolation from umbilical cord blood, mixed lymphocyte reaction

문서 내 토픽

1. CD34+ HSC isolation from Umbilical Cord Blood

HSC(Hematopoietic Stem Cell)는 인체 내에 존재하는 모든 혈액 세포로 분화가 가능한 줄기세포입니다. CD34 gene은 HSC부터 어느정도 분화가 이루어진 progenitor level에서까지 발현되기 때문에 이를 CD34+ cell들을 세포막에 붙어있는 CD34 protein의 특징을 이용해서 정제할 수 있습니다. 이 실험에서는 MACS microbead sorting 방법을 이용하여 항원항체반응을 통해 CD34+ HSC를 분리해내는 과정을 설명하고 있습니다.
2. Mixed Lymphocyte Reaction (MLR)

MLR은 T cell이 일정기간동안 특정물질이나 세포와 공동 배양 되었을 때 증식이 어느정도 이루어지는지 그 정도를 정량화하는 방법으로, 면역 반응이 억제되는 정도를 평가하는 척도로 사용됩니다. 실험에서는 Anti-CD3/28 bead와 IL-2를 이용하여 T cell을 활성화시키고, CFSE를 이용하여 T cell의 증식 정도를 측정하는 방법을 설명하고 있습니다.
3. CD34 protein

CD34는 transmembrane phosphoglycoprotein으로, 세포 막에서 cell-cell adhesion에 관여하는 factor입니다. CD34를 발현하는 세포들을 CD34+ cell이라고 부르며, 주로 umbilical cord나 bone marrow에서 발견됩니다. CD34는 HSC부터 어느정도 분화가 이루어진 progenitor level에서까지 발현되기 때문에, 이를 이용하여 HSC를 정제할 수 있습니다.
4. CFSE (Carboxyfluorescein succinimidyl ester)

CFSE는 세포막을 통과할 수 있고 세포 내부의 lysin residues와 결합하여 형광을 띠게 됩니다. 이 형광은 오랫동안 세포 내에 남아있으며, 세포가 분열할 때 딸세포에도 나누어져 들어가게 됩니다. 이를 이용하여 lymphocyte의 proliferation을 모니터링할 수 있습니다. 다만 높은 농도에서는 세포에 독성을 줄 수 있다는 단점이 있습니다.
5. Antibody-coupled magnetic beads

항체는 특정 항원에 대해 높은 특이성을 가지고 있습니다. 이를 이용하여 항체를 자기장에 반응하는 magnetic bead에 결합시키면, 세포 표면의 단백질과 결합한 bead를 자석을 이용해 분리해낼 수 있습니다. 이때 항체와 bead는 covalent attachment를 통해 비가역적으로 결합됩니다.

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1. CD34+ HSC isolation from Umbilical Cord Blood

CD34+ hematopoietic stem cells (HSCs) isolated from umbilical cord blood (UCB) are an important source of stem cells for regenerative medicine and cell-based therapies. The isolation of these cells is a critical step in the development of such therapies. The CD34 antigen is a well-established marker for identifying and isolating HSCs from various sources, including UCB. The process of CD34+ HSC isolation from UCB typically involves density gradient centrifugation to enrich for mononuclear cells, followed by immunomagnetic separation or flow cytometry to specifically isolate the CD34+ cell population. This allows for the purification and expansion of these cells for further research and clinical applications. The ability to efficiently and reliably isolate CD34+ HSCs from UCB is an important advancement in the field of stem cell biology and regenerative medicine.
2. Mixed Lymphocyte Reaction (MLR)

The Mixed Lymphocyte Reaction (MLR) is a widely used in vitro assay to assess the alloreactive potential of T cells. In this assay, T cells from one individual (responder) are co-cultured with antigen-presenting cells (APCs) from another individual (stimulator), typically with major histocompatibility complex (MHC) differences. This interaction triggers the activation and proliferation of the responder T cells, which can be measured by various methods, such as thymidine incorporation or flow cytometry. The MLR is a valuable tool for evaluating the immunogenicity of cells or tissues, as well as for studying the mechanisms of allorecognition and T cell-mediated immune responses. It has applications in transplantation immunology, autoimmunity, and the development of cell-based therapies. The MLR provides important insights into the complex interplay between the immune system and allogeneic cells, which is crucial for understanding and improving the success of various clinical interventions.
3. CD34 protein

The CD34 protein is a transmembrane glycoprotein that is widely used as a marker for hematopoietic stem and progenitor cells (HSPCs). CD34 is expressed on the surface of various cell types, including HSPCs, endothelial progenitor cells, and certain types of leukemia cells. The CD34 antigen plays a crucial role in the regulation of cell adhesion, migration, and differentiation, making it an important target for the isolation and characterization of HSPCs. The expression of CD34 is often used to identify and enrich for HSPCs from various sources, such as bone marrow, peripheral blood, and umbilical cord blood, for use in cell-based therapies and regenerative medicine applications. Additionally, the CD34 marker is valuable for the diagnosis and monitoring of hematological malignancies, as its expression patterns can provide important information about the disease state and response to treatment. Overall, the CD34 protein is a widely recognized and extensively studied marker that has become an essential tool in the field of stem cell biology and clinical hematology.
4. CFSE (Carboxyfluorescein succinimidyl ester)

CFSE (Carboxyfluorescein succinimidyl ester) is a fluorescent dye that is widely used in cell biology and immunology research to track cell division and proliferation. CFSE is a cell-permeant dye that binds to intracellular proteins, and it is equally distributed between daughter cells during cell division. This allows for the visualization and quantification of cell division by flow cytometry, as the fluorescence intensity of CFSE-labeled cells decreases with each cell division. The CFSE assay is particularly useful for studying the proliferative responses of various cell types, such as T cells, B cells, and stem cells, in response to different stimuli or environmental conditions. It has applications in areas such as immunology, stem cell biology, and cancer research, where understanding the dynamics of cell division and proliferation is crucial for understanding disease pathogenesis and developing effective therapies. The CFSE assay is a powerful and versatile tool that provides valuable insights into the complex cellular processes underlying immune responses, tissue regeneration, and disease progression.
5. Antibody-coupled magnetic beads

Antibody-coupled magnetic beads are a widely used tool in cell biology and biotechnology for the isolation, purification, and enrichment of specific cell populations or biomolecules. These beads are typically composed of a magnetic core coated with a polymer matrix, to which specific antibodies or other ligands are covalently attached. When a cell suspension or a sample containing the target molecule is incubated with the antibody-coupled magnetic beads, the target cells or molecules bind to the immobilized antibodies. The magnetic properties of the beads allow for the separation and isolation of the target cells or molecules using a magnetic field, without the need for complex and time-consuming centrifugation or other separation techniques. This method is highly versatile and can be used to isolate a wide range of cell types, including stem cells, immune cells, and cancer cells, as well as specific proteins, nucleic acids, and other biomolecules. The use of antibody-coupled magnetic beads has become an essential tool in various fields, such as cell therapy, diagnostics, and biotechnology, due to its efficiency, specificity, and ease of use.