Cryopreservation of living cells: principles and practice.

Increasingly, the cryopreservation of living cells is being attempted by researchers whose primary interest and experience is with the medical applications of those cells or tissues and whose prior experience with cryobiology may be negligible. It is therefore generally necessary to imitate some regimen used by others, perhaps with some other cell type and attempt to optimize the recovery empirically. This article makes no attempt to provide specific protocols for the many individual cell types.

Storage of red blood cells with improved maintenance of 2,3-bisphosphoglycerate.

Background: During storage, red blood cells (RBCs) rapidly lose 2,3-bisphosphoglycerate (2,3-DPG) leading to an increase in the affinity for O(2) and a temporary impairment of O(2) transport. Recent clinical evaluations indicate that the quality of transfused RBCs may be more important for patient survival than previously recognized.

Study Design And Methods: Glucose-free additive solutions (ASs) were prepared with sodium citrate, sodium gluconate, adenine, mannitol, and phosphates at high pH, a solution that can be heat-sterilized.

Red blood cells intended for transfusion: quality criteria revisited.

Great variation exists with respect to viability and function of fresh and stored red blood cells (RBCs) as well as of the contents of RBC hemoglobin (Hb) in individual units. Improved technology is available for the preparation as well as the storage of RBCs. The authors raise the question whether it may be time to revise current standards for RBC units.

Depletion of CD25+ cells from human T-cell enriched fraction eliminates immunodominance during priming with dendritic cells genetically modified to express a secreted protein.

The ability of dendritic cells (DCs), genetically modified with one of two types of plasmid DNA vaccines to stimulate lymphocytes from normal human donors and to generate antigen-specific responses, is compared. The first type, also called "secreted" vaccine (sVac), encodes for the full length of the human prostate-specific antigen (PSA) with a signal peptide sequence so that the expressed product is glycosylated and directed to the secretory pathway. The second type, truncated vaccines (tVacs), encodes for either hPSA or human prostate acidic phosphatase (hPAP), both of which lack signal peptide sequences and are retained in the cytosol and degraded by the proteasomes following expression.

Human dendritic cells genetically engineered to express cytosolically retained fragment of prostate-specific membrane antigen prime cytotoxic T-cell responses to multiple epitopes.

The ability of two plasmid DNA vaccines to stimulate lymphocytes from normal human donors and to generate antigen-specific responses is demonstrated. The first vaccine (truncated; tPSMA) encodes for only the extracellular domain of prostate-specific membrane antigen (PSMA). The product, expressed following transfection with this vector, is retained in the cytosol and degraded by the proteasomes.

Use of hollow fiber membrane filtration for the removal of DMSO from platelet concentrates.

It has been hypothesized that, in addition to freezing injury, some damage to platelets may result from the cell packing that occurs during removal of the cryoprotectant. This study examined DMSO removal by fluid exchange across hollow-fiber (HF) filters as an alternative to centrifugation. The DMSO solution with or without cell suspension was passed once through the filter.

WBC reduction in cryopreserved RBC units.

Background: WBC reduction of blood components by filtration is widely practiced to decrease the incidence of alloimmunization. Freezing RBCs reduces the WBC load but is insufficient to achieve the currently recommended US limit of 5 x 10(6) cells per unit.

Study Design And Methods: Blood units were WBC reduced by filtration or by buffy-coat (BC) removal and then frozen in the presence of a high-glycerol concentration.

In vivo transfection and/or cross-priming of dendritic cells following DNA and adenoviral immunizations for immunotherapy of cancer--changes in peripheral mononuclear subsets and intracellular IL-4 and IFN-gamma lymphokine profile.

In order to provoke an immune response, a tumor vaccine should not only maximize antigen-specific signals, but should also provide the necessary "co-stimulatory" environment. One approach is to genetically manipulate tumor cells to either secrete lymphokines (GM-CSF, IL-12, IL-15) or express membrane bound molecules (CD80, CD86). Furthermore, patient dendritic cells can be loaded with tumor-associated antigens or peptides derived from them and used for immunotherapy.

Naked DNA and adenoviral immunizations for immunotherapy of prostate cancer: a phase I/II clinical trial.

Introduction And Objectives: Animal studies have indicated that the use of syngeneic dendritic cells that have been transfected ex vivo with DNA for tumor-specific antigen results in tumor regression and decreased number of metastases. Additional studies have also suggested the possibility to modulate the dendritic cells in vivo either by 'naked' DNA immunization or by injecting replication-deficient viral vectors that carry the tumor-specific DNA. Using the prostate- specific membrane antigen (PSMA) as a target molecule, we have initiated a clinical trial for immunotherapy of prostate cancer.

Fas and Fas ligand expression on human peripheral blood leukocytes.

Objectives: Study of Fas and Fas ligand (Fas-L) expression, as well as sFas-L release, by fresh human peripheral blood leukocytes.

Methods: Flow cytometry, cytotoxicity, immunofluorescence staining of fresh smears. Western blotting.

Mechanisms of alloimmunization and immunosuppression by blood transfusions in an inbred rodent model.

During refrigerated storage leukocytes in donor blood progressively undergo apoptosis followed by secondary necrosis. Using an inbred rodent transfusion model, recipient animals received viable, necrotic, or apoptotic cells. While transfusion of viable blood MNCs stimulated production of IgM, IgG1 (Th2 type) and IgG2a (Th1-type) antidonor antibodies, leading to a suppression of subsequent DTH to donor antigens, transfusion of apoptotic donor cells led to neither alloimmunization nor immunosuppression.

Blood transfusion, blood storage and immunomodulation.

Allogeneic blood transfusion is the most frequent allotransplantation procedure performed on a routine basis with no prior HLA-typing. Roughly 50% of the recipients of unprocessed red cells and platelets become alloimmunized. Evidence also exists for some degree of transfusion-induced immunosuppression.

Blood transfusion and immunomodulation: a possible mechanism.

To induce an immunogenic response in vivo, an antigen-presenting (stimulator) cell must present both antigen-specific (class II MHC) and an accessory signal to the CD4 T cell. Failure to express the accessory signal has been shown in vitro to induce a state of specific unresponsiveness (anergy) in the T cell. We have shown that although stimulator cells in blood continue to express class II MHC molecules during refrigerated storage, their ability to present the accessory signal diminishes, reaching zero in all units tested by about 13 days.

Manipulating red cell intra- and extracellular pH by washing.

Washing red cells with solutions containing no anions capable of entering the cells is known to result in the loss of intracellular chloride and a counterflow of OH- which raises intracellular and lowers extracellular pH. Elevating the intracellular pH improves the quality of the cells during 4 degrees C storage. The extent to which intracellular pH can be raised and extracellular pH reduced depends not only on the inability of extracellular anions to enter the cells but also on the buffering capacity of the wash solution.

Refrigerated storage of washed red cells.

Red cells washed and stored in a citrate-phosphate-glucose-adenine solution at pH 7.4-7.6 demonstrate excellent maintenance of adenosine triphosphate, elevation of 2,3-diphosphoglycerate well above normal levels for more than 6 weeks, reduced hemolysis and 24-hour in vivo survival comparable to that of cells stored in ADSOL.

Cryoprotectant toxicity and cryoprotectant toxicity reduction: in search of molecular mechanisms.

Cryoprotectant toxicity is a fundamental obstacle to the full potential of artificial cryoprotection, yet it remains in general a poorly understood phenomenon. Unfortunately, most relevant biochemical studies to date have not met the basic criteria required for demonstrating mechanisms of toxicity. A model biochemical study of cryoprotectant toxicity was that of Baxter and Lathe, which demonstrated that alteration of a specific enzyme (fructose diphosphatase, or FDPase) was the cause of impaired glycolysis after treatment with and removal of dimethyl sulfoxide (D).

Costimulatory signals necessary for induction of T cell proliferation.

Two separate signals are required for induction of T cell proliferation. In an attempt to identify them we used polyclonal T cell activation with Con A, which requires costimulation with autologous accessory cells. The costimulatory activity is not constitutively expressed on accessory cells since such cells fixed immediately after separation from whole blood are unable to provide the necessary signal(s), although such activity is readily expressed after activation by incubation and such cells subsequently fixed will support Con A-induced T cell proliferation.

Induction of primary mixed leukocyte reactions with ultraviolet B or chemically modified stimulator cells.

Treatment of stimulator cells with 0.1% paraformaldehyde for 60 sec or ultraviolet-B (UV-B) irradiation (1000 J/m2) eliminates their ability to elicit T cell proliferation in a primary mixed leukocyte reaction. However, a T cell response equal to 20-40% of control value could be elicited by paraformaldehyde fixed or UV-B irradiated cells providing the latter are incubated at 37 degrees C for 18 hr prior to treatment.