Porcine Red Blood Cells Research Articles

Cryopreserved red blood cells maintain allosteric control of oxygen binding when utilizing trehalose as a cryoprotectant

One of the most common life-saving medical procedures is a red blood cell (RBC) transfusion. Unfortunately, RBCs for transfusion have a limited shelf life after donation due to detrimental storage effects on their morphological and biochemical properties. Inspired by nature, a biomimetics approach was developed to preserve RBCs for long-term storage using compounds found in animals with a natural propensity to survive in a frozen or desiccated state for decades. Trehalose was employed as a cryoprotective agent and added to the extracellular freezing solution of porcine RBCs. Slow cooling (−1 °C min−1) resulted in almost complete hemolysis (1 ± 1 % RBC recovery), and rapid cooling rates had to be used to achieve satisfactory cryopreservation outcomes. After rapid cooling, the highest percentage of RBC recovery was obtained by plunging in liquid nitrogen and thawing at 55 °C, using a cryopreservation solution containing 300 mM trehalose. Under these conditions, 88 ± 8 % of processed RBCs were recovered and retained hemoglobin (14 ± 2 % hemolysis). Hemoglobin's oxygen-binding properties of cryopreserved RBCs were not significantly different to unfrozen controls and was allosterically regulated by 2,3-bisphosphoglycerate. These data indicate the feasibility of using trehalose instead of glycerol as a cryoprotective compound for RBCs. In contrast to glycerol, trehalose-preserved RBCs can potentially be transfused without time-consuming washing steps, which significantly facilitates the usage of cryopreserved transfusible units in trauma situations when time is of the essence.

Initial investigation on the feasibility of porcine red blood cells from genetically modified pigs as an alternative to human red blood cells for transfusion.

The decline in blood donation rates and the ongoing shortage of blood products pose significant challenges to medical societies. One potential solution is to use porcine red blood cells (pRBCs) from genetically modified pigs as an alternative to human red blood cells (hRBCs). However, adverse immunological reactions remain a significant obstacle to their use. This study aimed to evaluate the compatibility of diverse genetically modified pRBCs with human serum. We acquired human complement-competent serum, complement 7 (C7)-deficient serum, and hRBCs from all ABO blood types. Additionally, we used leftover clinical samples from health checkups for further evaluation. pRBCs were collected from wild-type (WT) and genetically modified pigs: triple knockout (TKO), quadruple KO (QKO), and TKO/hCD55.hCD39 knockin (hCD55.hCD39KI). The extent of C3 deposition on RBCs was measured using flow cytometry after incubation in C7-deficient serum diluted in Ca++-enriched or Ca++-depleted and Mg++-enriched buffers. The binding of immunoglobulin (Ig) M/IgG antibody to RBCs after incubation in ABO-type human serum was evaluated using flow cytometry. Naïve human serum- or sensitized monkey serum-mediated hemolysis was also evaluated. Phagocytosis was assessed by incubating labeled RBCs with the human monocytic cell line THP-1 and measurement by flow cytometry. All three genetic modifications significantly improved the compatibility of pRBCs with human serum relative to that of WT pRBCs. The extent of IgM/IgG binding to genetically modified pRBCs was lower than that of WT pRBCs and similar to that of O-type hRBCs. Total and alternative pathway complement activation in all three genetically modified pRBCs was significantly weaker than that in WT pRBCs and did not differ from that in O-type hRBCs. The extent of serum-mediated hemolysis and phagocytosis of these genetically modified pRBCs was low and similar to that of O-type hRBCs. Sensitized monkey serum-mediated hemolysis in QKO and TKO/hCD55.hCD39KI pRBCs was higher than in O-type hRBCs but lower than in TKO pRBCs. The elimination of porcine carbohydrate antigens in genetically modified pigs significantly enhanced pRBC compatibility with naïve human sera, which was comparable to that of O-type hRBCs. These findings provide valuable insights into the development of pRBCs as potential alternatives to hRBCs.

P.45: Initial investigation on the feasibility of porcine red blood cells from genetically modified pigs as a human red blood cell alternative for transfusion

P.45: Initial investigation on the feasibility of porcine red blood cells from genetically modified pigs as a human red blood cell alternative for transfusion

41 Assessing the feasibility of genetically modified porcine red blood cells as an alternative to human red blood cells for transfusion

41 Assessing the feasibility of genetically modified porcine red blood cells as an alternative to human red blood cells for transfusion

Antibodies against hyaluronan oligosaccharides in xenotransplantation.

Carbohydrate-specific antibodies are significant mediators of xenograft rejection. This study analyzed the carbohydrate specificity of antibodies in baboons before and after xenotransplantation of organs or injection of porcine red blood cells from hDAF transgenic pigs, using a glycan array with structurally defined glycans. Antibodies against hyaluronic acid disaccharide (HA2) showed the highest reactivity at baseline and rose after xenogeneic exposure. We also investigated in the serum of baboons that underwent xenotransplantation with either hDAF or hDAF/hMCP transgenic pig organs and Lewis rats after hamster-skin xenotransplantation the specificity of anti-HA antibodies on a glycan microarray representing HA oligosaccharides containing from two to 40 saccharides. Notably, the HA oligosaccharides ranging from 32 to 40 saccharides exhibited the highest antibody binding intensities at baseline in baboon and rat sera. After xenotransplantation, antibodies against HA38 and HA40 in baboons, and HA32, HA34, and HA36 in rats showed the highest titer increases. The changes of anti-HA IgM and IgG antibodies in rats after skin xenotransplantation was also confirmed by an ELISA specific for HA2, HA24, and HA85 antibodies. Thus, xenotransplantation is associated with increased antibodies against HA-oligosaccharides, which may represent a new target for intervention.

Molecular Simulation Study on the Interaction between Porcine CR1-like and C3b.

The molecular basis of porcine red blood cell immune adhesion function stems from the complement receptor type 1-like (CR1-like) on its cell membrane. The ligand for CR1-like is C3b, which is produced by the cleavage of complement C3; however, the molecular mechanism of the immune adhesion of porcine erythrocytes is still unclear. Here, homology modeling was used to construct three-dimensional models of C3b and two fragments of CR1-like. An interaction model of C3b-CR1-like was constructed by molecular docking, and molecular structure optimization was achieved using molecular dynamics simulation. A simulated alanine mutation scan revealed that the amino acids Tyr761, Arg763, Phe765, Thr789, and Val873 of CR1-like SCR 12-14 and the amino acid residues Tyr1210, Asn1244, Val1249, Thr1253, Tyr1267, Val1322, and Val1339 of CR1-like SCR 19-21 are key residues involved in the interaction of porcine C3b with CR1-like. This study investigated the interaction between porcine CR1-like and C3b using molecular simulation to clarify the molecular mechanism of the immune adhesion of porcine erythrocytes.

Genetic modifications designed for xenotransplantation attenuate sialoadhesin-dependent binding of human erythrocytes to porcine macrophages.

The phenomenon of diminishing hematocrit after in vivo liver and lung xenotransplantation and during ex vivo liver xenoperfusion has largely been attributed to action by resident liver porcine macrophages, which bind and destroy human erythrocytes. Porcine sialoadhesin (siglec-1) was implicated previously in this interaction. This study examines the effect of porcine genetic modifications, including knockout of the CMAH gene responsible for expression of Neu5Gc sialic acid, on the adhesion of human red blood cells (RBCs) to porcine macrophages. Wild-type (WT) porcine macrophages and macrophages from several strains of genetically engineered pigs, including CMAH gene knockout and several human transgenes (TKO+hTg), were incubated with human RBCs and "rosettes" (≥3 erythrocytes bound to one macrophage) were quantified by microscopy. Our results show that TKO+hTg genetic modifications significantly reduced rosette formation. The monoclonal antibody 1F1, which blocks porcine sialoadhesin, significantly reduced rosette formation by WT and TKO+hTg macrophages compared with an isotype control antibody. Further, desialation of human RBCs with neuraminidase before addition to WT or TKO+hTg macrophages resulted in near-complete abrogation of rosette formation, to a level not significantly different from porcine RBC rosette formation on porcine macrophages. These observations are consistent with rosette formation being mediated by binding of sialic acid on human RBCs to sialoadhesin on porcine macrophages. In conclusion, the data predict that TKO+hTg genetic modifications, coupled with targeting of porcine sialoadhesin by the 1F1 mAb, will attenuate erythrocyte sequestration and anemia during ex vivo xenoperfusion and following in vivo liver, lung, and potentially other organ xenotransplantation.

Digestibility of amino acids in fish meal and blood-derived protein sources fed to pigs.

ObjectiveAn experiment was conducted to determine the standardized ileal digestibility (SID) of amino acids (AA) in fish meal (FM) and blood-derived protein sources including spray-dried porcine plasma (SDPP), porcine red blood cells (PRBC), and blood meal (BM) fed to growing pigs.MethodsTen barrows (mean initial body weight of 22.1±1.54 kg) surgically fitted with T-cannulas at the distal ileum were allotted to a duplicated 5×4 incomplete Latin square design with 5 experimental diets and 4 periods. Four experimental diets were prepared to contain FM, SDPP, PRBC, or BM as the sole source of nitrogen. A nitrogen-free diet was prepared and included to estimate the basal ileal endogenous losses of AA. For the 7-day experimental period, pigs were fed for 5 days as adaptation, and ileal digesta samples were collected for 9 hours on days 6 and 7.ResultsThe SID of crude protein in BM (48.0%) was less (p<0.05) than in FM, SDPP, and PRBC (83.4%, 83.9%, and 87.3%, respectively). Pigs fed the diet containing BM had less (p<0.05) SID of AA, except isoleucine and proline, than those fed the diet containing FM, SDPP, or PRBC. Among FM, SDPP, and PRBC, there was no difference in the SID of crude protein and all AA, except isoleucine. The SID of isoleucine in PRBC and BM (62.7% and 48.3%, respectively) was less (p<0.05) than in FM and SDPP (88.0% and 84.9%, respectively). The SID of lysine in FM, SDPP, PRBC, and BM was 85.4%, 84.9%, 89.7%, and 51.9%, respectively.ConclusionThe SID of most AA was not different among FM, SDPP, and PRBC, but BM had lower SID of most AA than FM, SDPP, and PRBC.

Comparison of injectable trace minerals vs. adjuvant on measures of innate and humoral immune responses of beef heifers

• Injectable trace minerals (ITM), adjuvant (ADJ) and saline did not impact calf growth • Haptoglobin and ceruloplasmin were greatest for ADJ but similar between SAL and ITM • ITM enhanced calf antibody titers but not to the extent as ADJ injection Previous studies reported heightened innate and humoral immune responses among cattle administered injectable trace minerals (ITM) concurrent to immunogen challenge, thus suggesting a potential adjuvant-like activity of ITM. To pursue this concept further, a two-year study was conducted at the Mountain Research Station (North Carolina State University, Waynesville) to evaluate immunological responses of Angus heifers (n = 22, 235 ± 18 kg of BW and 225 ± 4 d of age in yr 1; n = 36, 237 ± 6 kg of BW and 247 ± 5 d of age in yr 2). On d 0 (30 days after weaning), heifers were administered two 5-mL i.m. injections of porcine red blood cells (PRBC; Lampire Biological Laboratories, Pipersville, PA) solution (25% PRBC and 75% sterile PBS; Amresco, Solon, OH), concurrent to either a single 2.5-mL s.c. injection of sterile saline (SAL; 0.9% NaCl), a squalene-based oil-in-water adjuvant (ADJ; AddaVax; InvivoGen, Inc. San Diego, CA) that specifically induces a humoral immune response, or injectable trace minerals (ITM; MultiMin 90; MultiMin USA, Inc. Fort Collins, CO; 60, 10, 15 and 5 mg/mL of Zn, Mn, Cu and Se). During the 28-d study, heifers were offered tall fescue hay ( Lolium arundinacea ) and concentrate at 1.2 and 1.0% of BW (DM basis), respectively, blended with 114 g daily of a complete mineral mix. Shrunk BW was recorded on d 0 and 28 following 12 h of feed and water withdrawal. Blood samples were collected on d 0, 2, 7, 15, and 21 for determination of antibody titers against PRBC and plasma concentrations of ceruloplasmin and haptoglobin. Liver biopsy samples were collected 1 mo prior to weaning and on d 15 after weaning for determination of trace mineral status. No differences were detected ( P ≥ 0.13) for BW change, ADG, G:F, or DMI. Liver Se concentrations on d 15 were greater ( P ≤ 0.01) for heifers receiving ITM compared to SAL and ADJ treatments. Plasma haptoglobin concentrations on d 2 were greatest for ADJ ( P < 0.0001) and did not differ between SAL and ITM heifers ( P ≥ 0.46). Plasma ceruloplasmin concentrations on d 2 were greatest for ADJ ( P ≤ 0.04) and did not differ between SAL and ITM heifers ( P = 0.39). On d 7, plasma concentrations of ceruloplasmin did not differ ( P = 0.37) between ADJ and ITM, were less for SAL vs. ADJ ( P = 0.005), and tended to differ ( P = 0.064) between ITM and SAL heifers. Serum PRBC titers were greatest for ADJ ( P ≤ 0.03) vs. SAL on d 7, 15, and 21 and ITM on d 7 ( P < 0.001). Serum PRBC titers did not differ between SAL and ITM on d 7 and 15 ( P ≥ 0.23), but were greater in ITM vs. SAL on d 21 ( P = 0.03). In summary, ITM prolonged the antibody titers against PRBC on d 21 compared to saline injection in mineral-adequate beef calves, but not as quickly or to the extent of ADJ injection.

Modified haemagglutination inhibition assay for the detection of canine parvovirus type 2 antibodies in dog sera

Canine parvovirus type 2 (CPV-2) infection is associated with severe gastroenteritis in puppies. Quantification of CPV-2 specific antibodies before vaccination can reveal the presence of interfering maternal-derived immunity and facilitate timing of effective immunisation. Inhibition of haemagglutination (HI) is commonly used to measure CPV-2-specific antibody levels in serum. However, the presence of nonspecific agglutinins in canine serum and artefactual precipitation of red blood cells (RBC) are both limitations of the assay. In this study, we compared the standard HI protocol with a refined HI protocol, in which canine serum was pre-incubated with porcine RBC for 12 h to remove nonspecific agglutinins and a lower concentration (0.1% vs. 0.8%) of porcine RBC suspensions was used to limit artefactual precipitation of RBC. A panel of canine sera, collected from 80 dogs of different ages and with different neutralising antibody titres, was analysed.Nonspecific agglutinins were identified in most (97%) serum samples from puppies <4 months of age and in only 7% dogs 6 months old. Pre-treatment of serum samples was effective in removing nonspecific agglutinins from all samples and artefactual precipitation of RBCs was not noted when 0.1% RBC suspensions were used. Refinement of the HI protocol has increased the accuracy of interpretation and reduced the interference of nonspecific agglutinins, primarily seen in puppies. This reduces the likelihood of incorrect assessment of passive or active immunity in puppies when deciding whether to administer or defer vaccination, which could potentially leave them susceptible to CPV-2 infection.

Ionic Fluoropolyphosphazenes as Potential Adhesive Agents for Dental Restoration Applications

Clinically, the use of photo-activated polymer-based dental composites is the preferred method to restore carious teeth due to their esthetics and ease of application. However, the longevity of these resin-based composite tooth restorations can be compromised by the sensitivity of the bonded interface between the composite and the tooth surface. The objective of the current study was to modify the tooth surface with novel fluorinated polyphosphazenes (PPZs), thereby improving the stability of the interface. Binding isotherms of PPZs with collagen (CLG) and hydroxyapatite (HA), two of the primary components of teeth, were established and indicate significant and stable adsorption to the surfaces of these materials. PPZs were also shown to protect CLG against acidic dissolution in a model system. A composite material consisting of the fluorinated polymer and CLG demonstrated three-dimensional stability and significant hydrophobicity. Additionally, no hemolytic activity was observed when evaluated using a porcine red blood cells (RBC) assay. Bovine dentin treated with PPZs demonstrated increased contact angle (hydrophobicity) compared with control samples and resisted fluid penetration when assessed using a dye penetration study. Finally, microhardness evaluation of bovine dentin treated with PPZs and exposed to an acidic challenge showed that treated dentin resisted demineralization. The hardness of the untreated control was significantly reduced after exposure when compared with the PPZ-treated samples. This study represents a novel approach to overcoming the current limitations of composite restorations. These results are promising to improve the longevity of composite dental restorations and may have wider use in sealants, varnishes, and other dental applications. Tooth decay remains a prevalent problem worldwide. Polymer-based composites are the most frequently used tooth restorative used in the clinic. The longevity of these fillings is limited due to conditions in the mouth that can weaken the adhesive used to bond the composite to the natural tooth. The current study uses novel polyphosphazenes (PPZs), hybrid organic-inorganic macromolecules with tunable hydrophilic-hydrophobic properties to coat the tooth surface to achieve better compatibility with the adhesive, thereby improving the longevity of the restoration. Results indicate that PPZs have significant and stable adsorption onto teeth, which may lead to a more stable bonded interface.

Anthelminthic Activity of Assassin Bug Venom against the Blood Fluke Schistosoma mansoni.

Helminths such as the blood fluke Schistosoma mansoni represent a major global health challenge due to limited availability of drugs. Most anthelminthic drug candidates are derived from plants, whereas insect-derived compounds have received little attention. This includes venom from assassin bugs, which contains numerous bioactive compounds. Here, we investigated whether venom from the European predatory assassin bug Rhynocoris iracundus has antischistosomal activity. Venom concentrations of 10–50 µg/mL inhibited the motility and pairing of S. mansoni adult worms in vitro and their capacity to produce eggs. We used EdU-proliferation assays to measure the effect of venom against parasite stem cells, which are essential for survival and reproduction. We found that venom depleted proliferating stem cells in different tissues of the male parasite, including neoblasts in the parenchyma and gonadal stem cells. Certain insect venoms are known to lyse eukaryotic cells, thus limiting their therapeutic potential. We therefore carried out hemolytic activity assays using porcine red blood cells, revealing that the venom had no significant effect at a concentration of 43 µg/mL. The observed anthelminthic activity and absence of hemolytic side effects suggest that the components of R. iracundus venom should be investigated in more detail as potential antischistosomal leads.

Genetic parameter estimation and genome-wide association study (GWAS) of red blood cell count at three stages in a Duroc×Erhualian pig population

Red blood cells play an essential role in the immune system. Moreover, red blood cell count (RBC) is an important clinical indicator of various diseases, including anemia, type 2 diabetes and the metabolic syndrome. Thus, it is necessary to reveal the genetic mechanism of RBC for animal disease resistance breeding. However, quite a few studies had focused on porcine RBC, especially at different stages. Thus, studies on porcine RBC at different stages are needed for disease resistant breeding. In this study, the porcine RBC of 20-, 33-, and 80-day old were measured, and genetic parameter estimation and genome-wide association study (GWAS) were both performed. As a result, the heritability was about 0.6 at the early stages, much higher than that at 80 days. Nine novel genome wide significant single nucleotide polymorphisms (SNPs), located at Sus scrofa chromosome (SSC)3, 4, 8, 9, 10 and 15, respectively, were identified. Further, TGFβ2, TMCC2 and PPP1R15B genes were identified as important candidate genes of porcine red blood cell count. So different SNPs and candidate genes were found significantly associated with porcine RBC at different stages, suggesting that different genes might play key roles on porcine RBC at different stages. Overall, new evidences were offered in this study for the genetic bases of animal RBC, and that the SNPs and candidate genes would be useful for disease resistant breeding of pig.

130 Comparison of amino acid digestibility in fish meal, porcine plasma, porcine red blood cell, and blood meal between broiler chickens and young pigs

Abstract The objective of this study was to compare the standardized ileal digestibility (SID) of AA in fish meal (FM), porcine plasma (PP), porcine red blood cell (RBC), and blood meal (BM) between broiler chickens and pigs. Five diets were prepared for both species including 4 diets containing test ingredients and a nitrogen-free diet. In Exp. 1, 416 male broiler chickens (initial BW = 705 ± 100.0 g) at d 18 post hatching were assigned to diets in a randomized complete block design with BW as a blocking factor. After 5 d of feeding, birds were euthanized for collection of ileal digesta. In Exp. 2, 10 barrows (initial BW = 22.1 ± 1.54 kg) surgically fitted with T-cannulas at the distal ileum were allotted to duplicate 5 × 4 incomplete Latin square design with 5 diets and 4 periods. Each experimental period consisted of 5-d adaptation and 2-d ileal digesta collection periods. Data for Exp. 1 and 2 were considered together as a 2 × 4 factorial arrangement with the effects of species (i.e., broiler chickens and pigs) and four diets (i.e., FM, PP, RBC, and BM). There were interactions (P &amp;lt; 0.05) between species and diets on the SID of AA except for Pro. The SID of Lys, Met, Thr, and Trp in FM, PP, and RBC for broiler chickens were not different from values for pigs; however, the SID of respective AA in BM for broiler chickens were less than values for pigs. The SID of indispensable AA in BM were less than in FM, PP, or RBC within each species, but the SID of indispensable AA in BM for broiler chickens was less than values for pigs. In conclusion, utilization of AA in animal protein ingredients containing high concentration of CP varies between broiler chickens and pigs.

New Family of Water-Soluble Sulfo-Fluoro Polyphosphazenes and Their Assembly within Hemocompatible Nanocoatings.

In this work, novel sulfo-fluoro polyphosphazenes (PPzs) were synthesized via macromolecular substitution of polydichlorophosphazene utilizing "non-covalent protection" methodology by converting acid functionalities into hydrophobic alkylammonium salts. Resulting PPzs showed excellent solubility in aqueous solutions over a broad pH range and contained ∼25% sulfo- groups and 20% either trifluoroethoxy- (FESP) or trifluoromethylphenoxy- (FPSP) side groups, as determined by NMR spectroscopy. Their polyelectrolyte behavior was evaluated by binding with an oppositely charged polyion, branched polyethylenimine (PEI), which resulted in the formation of interpolymer complexes as shown by dynamic light scattering (DLS). Contrary to a sulfonated, nonfluorinated PPz homopolymer (SP), fluorinated macromolecules effectively bound human serum albumin (HSA) as revealed by dynamic light scattering and asymmetric flow field flow fractionation (AF4) studies. Moreover, FESP and FPSP both displayed low hemolytic activity as evaluated in solution using porcine red blood cells. Using the layer-by-layer (LbL) technique, FESP and FPSP were assembled into nanocoatings with PEI. Both fluorinated and nonfluorinated sulfo PPzs showed linear growth with PEI because of strong ionic pairing between sulfo and amino groups. However, films of fluorinated PPzs displayed higher hydrophobicity, lower swelling, and improved stability in high ionic strength environment when compared to coatings formed by a sulfonated, nonfluorinated SP, or a carbon-chain polymer poly(styrene sulfonic acid). Hemocompatibility of FESP and FPSP nanofilms was demonstrated in vitro using whole rabbit blood hemolysis tests, which showed less than 1% hemolysis. Altogether, the present study introduces a new class of hemocompatible, sulfo-fluoropolymers that shows promise for life science applications.

Biomimetic Glyconanoparticle Vaccine for Cancer Immunotherapy.

Cancer immunotherapy aims to harness the immune system to combat malignant processes. Transformed cells harbor diverse modifications that lead to formation of neoantigens, including aberrantly expressed cell surface carbohydrates. Targeting tumor-associated carbohydrate antigens (TACA) hold great potential for cancer immunotherapy. N-glycolylneuraminic acid (Neu5Gc) is a dietary non-human immunogenic carbohydrate that accumulates on human cancer cells, thereby generating neoantigens. In mice, passive immunotherapy with anti-Neu5Gc antibodies inhibits growth of Neu5Gc-positive tumors. Here, we designed an active cancer vaccine immunotherapy strategy to target Neu5Gc-positive tumors. We generated biomimetic glyconanoparticles using engineered αGal knockout porcine red blood cells to form nanoghosts (NGs) that either express (NGpos) or lack expression (NGneg) of Neu5Gc-glycoconjugates in their natural context. We demonstrated that optimized immunization of "human-like" Neu5Gc-deficient Cmah-/- mice with NGpos glyconanoparticles induce a strong, diverse and persistent anti-Neu5Gc IgG immune response. The resulting anti-Neu5Gc IgG antibodies were also detected within Neu5Gc-positive tumors and inhibited tumor growth in vivo. Using detailed glycan microarray analysis, we further demonstrate that the kinetics and quality of the immune responses influence the efficacy of the vaccine. These findings reinforce the potential of TACA neoantigens and the dietary non-human sialic acid Neu5Gc, in particular, as immunotherapy targets.

Porcine signal regulatory protein alpha binds to human CD47 to inhibit phagocytosis: Implications for human hematopoietic stem cell transplantation into severe combined immunodeficient pigs.

Severe combined immunodeficient (SCID) pigs are an emerging animal model being developed for biomedical and regenerative medicine research. SCID pigs can successfully engraft human-induced pluripotent stem cells and cancer cell lines. The development of a humanized SCID pig through xenotransplantation of human hematopoietic stem cells (HSCs) would be a further demonstration of the value of such a large animal SCID model. Xenotransplantation success with HSCs into non-obese diabetic (NOD)-derived SCID mice is dependent on the ability of NOD mouse signal regulatory protein alpha (SIRPA) to bind human CD47, inducing higher phagocytic tolerance than other mouse strains. Therefore, we investigated whether porcine SIRPA binds human CD47 in the context of developing a humanized SCID pig. Peripheral blood mononuclear cells (PBMCs) were collected from SCID and non-SCID pigs. Flow cytometry was used to assess whether porcine monocytes could bind to human CD47. Porcine monocytes were isolated from PBMCs and were subjected to phagocytosis assays with pig, human, and mouse red blood cell (RBC) targets. Blocking phagocytosis assays were performed by incubating human RBCs with anti-human CD47 blocking antibody B6H12, non-blocking antibody 2D3, and nonspecific IgG1 antibody and exposing to human or porcine monocytes. We found that porcine SIRPA binds to human CD47 in vitro by flow cytometric assays. Additionally, phagocytosis assays were performed, and we found that porcine monocytes phagocytose human and porcine RBCs at significantly lower levels than mouse RBCs. When human RBCs were preincubated with CD47 antibodies B6H12 or 2D3, phagocytosis was induced only after B6H12 incubation, indicating the lower phagocytic activity of porcine monocytes with human cells requires interaction between porcine SIRPA and human CD47. We have shown the first evidence that porcine monocytes can bind to human CD47 and are phagocytically tolerant to human cells, suggesting that porcine SCID models have the potential to support engraftment of human HSCs.

Porcine SIRPA binds to human CD47 to inhibit phagocytosis: Implications for human hematopoietic stem cell transplantation into severe combined immunodeficient (SCID) pigs

Abstract Xenotransplantation success with human hematopoietic stem cells into non-obese diabetic (NOD)-derived severe combined immunodeficient (SCID) mice is dependent on the ability of NOD mouse signal regulatory protein alpha (SIRPA) to bind human CD47 (hCD47). Interaction between NOD mouse SIRPA and hCD47 results in higher phagocytic tolerance compared to other SCID mouse strains. In the context of developing a humanized SCID pig, we investigated whether porcine SIRPA binds hCD47 and subsequently inhibit phagocytosis of human cells. Porcine SIRPA bound to hCD47 in vitro. Porcine monocytes phagocytosed human and porcine red blood cells (RBCs) at significantly lower levels than mouse RBCs. When human RBCs were pre-incubated with CD47 blocking antibody B6H12 or non-blocking antibody 2D3, phagocytosis was induced only after B6H12 incubation, indicating the lower phagocytic activity requires interaction between porcine SIRPA and hCD47. Collectively, we provide the first evidence that porcine monocytes may be phagocytically tolerant towards human cells, suggesting that porcine SCID models have the potential to support engraftment of human stem cells.

Unloading of cryoprotectants from cryoprotectant-loaded cells on a microfluidic platform.

In this paper, a multistep dilution-filtration microdevice (MDFD) is developed for unloading cryoprotectants from cryoprotectant-loaded cells. The MDFD contained a diluent producing region, a dilution-filtration execution region, and a filtrate collection region. It was made of two patterned PMMA stamps with four pieces of sandwiched PVDF membranes. Firstly, the performances of the mixers that were used in the diluent producing region and the dilution-filtration execution region were assessed using fluorescence experiments. Then, the effect of the MDFD structure on the loss of cells was investigated by applying the MDFD to unload glycerin from glycerin-loaded porcine red blood cells. Finally, the effects of the cell density, glycerin concentration, and membrane pore size on the clearance efficiency of glycerin (C G ), the survival rate of cells (S C ) and the recovery rate of cells (R C ) have been studied. Under the designed conditions, C G achieved ~80% and S C reached ~90%. However, R C was only ~40%, mainly resulting from the cells detained on the membrane surface and squeezed through the membrane pores into the filtrate. Increasing the membrane pore size caused high C G and S C , but low R C . For a low glycerin concentration, C G , S C , and R C were all high. For a high cell density, C G was high, but both S C and R C were low. This work is of significance to develop a microfluidic chip for unloading cryoprotectants from a small amount of cryopreserved cell samples.

THE EFFECTS OF EXTENSIONAL STRESS ON RED BLOOD CELL HEMOLYSIS

Artificial prostheses create non-physiologic flow conditions with stress forces that may induce blood cell damage, particularly hemolysis. Earlier computational fluid dynamics (CFD) prediction models based on a quantified power model showed significant discrepancies with actual hemolysis experiments. These models used the premise that shear stresses act as the primary force behind hemolysis. However, additional studies have suggested that extensional stresses play a more substantial role than previously thought and should be taken into account in hemolysis models. We compared extensional and shear stress flow fields within the contraction of a short capillary with sharp versus tapered entrances. The flow field was calculated with CFD to determine stress values, and hemolysis experiments with porcine red blood cells were performed to correlate the effects of extensional and shear stress on hemolysis. Our results support extensional stress as the primary mechanical force involved in hemolysis, with a threshold value of 1000 Pa under exposure time less than 0.060 ms.