ABSTRACT
Surface modification of cationic lipoplexes has been carried out by means of a postgrafting reaction. The original lipoplexes described comprise a cationic lipid, a neutral lipid, poly(ethylene glycol)-cholesterol (with or without a targeting ligand) and DNA. Modifying their surface via a chemical, postgrafting reaction did not alter their size (approximately 100 nm) nor their ability to compact DNA, but did give a reduced zeta potential (approximately 0 mV) to afford surface neutral particles. With the modified lipoplexes nonspecific NIH3T3 cell surface binding in vitro was inhibited. Intravenous injection of the neutralized lipoplexes in mice showed decreased accumulation of the particles in the lung as compared to PEGylated cationic lipoplexes. Tumor targeting was also achieved in vivo by the addition of an RGD-PEG-Cholesterol as a lipid-ligand in the postgrafted lipoplex formulation.
Subject(s)
Colloids/chemistry , DNA/administration & dosage , Gene Transfer Techniques/instrumentation , Acetates/chemistry , Acetylation , Animals , Cations/chemistry , Colloids/pharmacokinetics , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , DNA/genetics , Female , Genes, Reporter/genetics , Lipids/chemistry , Liposomes , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Mice , Mice, Inbred C57BL , Microscopy, Electron, Transmission , Molecular Structure , NIH 3T3 Cells , Oligopeptides/chemistry , Polyethylene Glycols/chemistry , Sulfates/chemistryABSTRACT
We have established rapid procedures that negatively deplete and positively select for specific murine cell populations. By using polystyrene tissue culture flasks containing a covalently bound mouse anti-rat antibody and specific anti-mouse, cell-surface antigen antibodies, we easily and efficiently depleted greater than 90% of the mature lineage cells from murine bone marrow. This selection procedure resulted in an enrichment of progenitor colonies (CFU-Cs) in murine bone marrow. Using the same polystyrene tissue culture devices, we can directly isolate CD117+ (c-kit+) murine hematopoietic cells. As few as 2000 of these CD117+ cells rescued and reconstituted lethally irradiated recipients in a murine bone marrow transplant model.
Subject(s)
Cell Separation/methods , Culture Techniques/methods , Hematopoietic Stem Cells , Animals , Bone Marrow Transplantation , Cell Lineage , Culture Techniques/instrumentation , Flow Cytometry/methods , Lymphocytes/immunology , Mice , Mice, Inbred C57BL , Proto-Oncogene Proteins c-kit/immunologyABSTRACT
Realization of the full potential of human umbilical cord blood (HUCB) as a source of transplantable hematopoietic progenitor cells (HPC) will be contingent on the development of a reliable method for ex vivo cell processing and stem cell enrichment. A first step in stem cell enrichment protocols involves depletion of the red blood cells (RBC) in the sample. We have compared several RBC depletion methods on the basis of recovery of total nucleated cells. It was found that 3% gelatin was effective at depleting RBC with a nucleated cell recovery of 72.4 +/- 7.3% (mean +/- standard deviation [SD]) in the HUCB samples. Several lectins were also used for processing HUCB and compared for their ability to remove RBC. Of these, soybean agglutinin (SBA) was found to remove RBC without substantial loss of HPC. Moreover, sequential treatment of HUCB with 3% gelatin and the AIS MicroCELLector SBA resulted in a product with < 1% hematocrit, 57% reduction in T cells, 35% nucleated cell recovery, and relatively high yields of burst-forming units-erythroid (BFU-E) (61%) and colony-forming units-granulocyte/macrophage (CFU-GM) (58%) and -mixed (CFU-GEMM) (66%). In a separate series of studies, enrichment of the CD34+ subset in HUCB was accomplished by processing HUCB with Ficoll followed by sequential treatment with the AIS MicroCELLector SBA and AIS MicroCELLector CD34. The CD34+ fraction was enriched for BFU-E (14-fold), CFU-GM (nine-fold), and CFU-GEMM (five-fold) with an overall purity ¿ > or = 92% CD34+ by flow cytometry. This report demonstrates that 3% gelatin and the AIS MicroCELLector SBA can be combined as an ex vivo processing technique to reduce the volume of the HUCB product by depleting RBC and T cells while still maintaining a high recovery of HPC. Moreover, separation of highly enriched CD34+ cells from HUCB is achievable and opens up the possibility of using CD34+ cells from HUCB for ex vivo progenitor cell expansion for transplantation, transfusion, and gene therapy.
Subject(s)
Antigens, CD/analysis , Cell Separation/methods , Erythrocytes , Fetal Blood/cytology , Hematopoietic Stem Cells/cytology , Lectins , Plant Lectins , Soybean Proteins , Antigens, CD34 , Centrifugation, Density Gradient , Colony-Forming Units Assay , Female , Flow Cytometry , Gelatin , Hematopoietic Stem Cells/immunology , Humans , Immunophenotyping , Lectins/metabolism , PregnancyABSTRACT
Human umbilical cord blood (HUCB) is a rich source of hematopoietic progenitor cells (HPC). Nevertheless, it has been previously reported that attempts to enrich for HPC resulted in substantial loss of progenitor cells. We have developed a cell processing technique for HUCB using 3% gelatin sedimentation and the AIS MicroCELLectorSBA and CD34. By this technique, we were able to deplete red blood cells (RBC) and obtain a partial T cell depletion, while maintaining 70-90% of the HPC. The results indicate that it may be possible to manipulate cord blood without significant loss of HPC, thus enhancing its utility for transplantation.
