Larry C. Lasky

Effects of Filtration Seeding on Cell Density, Spatial Distribution, and Proliferation in Nonwoven Fibrous Matrices

The cell seeding density and spatial distribution in a 3‐D scaffold are critical to the morphogenetic development of an engineered tissue. A dynamic depth‐filtration seeding method was developed to improve the initial cell seeding density and spatial distribution in 3‐D nonwoven fibrous matrices commonly used as tissue scaffolds. In this work, trophoblast‐like ED27 cells were seeded in poly(ethylene terephthalate) (PET) matrices with various porosities (0.85−0.93). The effects of the initial concentration of cells in the suspension used to seed the PET matrix and the pore size of the matrix on the resulting seeding density and subsequent cell proliferation and tissue development were studied. Compared to the conventional static seeding method, the dynamic depth‐filtration seeding method gave a significantly higher initial seeding density (2−4 × 107 vs 4 × 106 cells/cm3), more uniform cell distribution, and a higher final cell density in the tissue scaffold. The more uniform initial cell spatial distribution from the filtration seeding method also led to more cells in S phase and a prolonged proliferation period. However, both uniform spatial cell distribution and the pore size of the matrices are important to cell proliferation and morphological development in the seeded tissue scaffold. Large‐pore matrices led to the formation of cell aggregates and thus might reduce cell proliferation. The dynamic depth‐filtration seeding method is better in providing a higher initial seeding density and more uniform cell distribution and is easier to apply to large tissue scaffolds. A depth‐filtration model was also developed and can be used to simulate the seeding process and to predict the maximum initial seeding densities in matrices with different porosities.

An instrument to determine the magnetophoretic mobility of labeled, biological cells and paramagnetic particles

Abstract An instrument is described and discussed which can determine the magnetophoretic mobility of immunomagnetically labeled cells and paramagnetic particles. Through the use of a well-characterized magnetic energy gradient and a computer algorithm, cell tracking velocimetry, it is possible to obtain the mean and distribution of the magnetophoretic mobility for samples with greater than 10 3 individuals.

Human cord cell hematopoiesis in three-dimensional nonwoven fibrous matrices: in vitro simulation of the marrow microenvironment.

Current hematopoietic culture systems mainly utilize two-dimensional devices with limited ability to promote self-renewal of early progenitors. In vivo-like three-dimensional (3-D) culture environments might be conducive to regulating stem cell proliferation and differentiation similar to in vivo hematopoiesis. The few 3-D cultures reported in the literature either produced few progenitors or provided little information about microenvironment. In this study, we constructed a 3-D hematopoietic microenvironment composed of nonwoven matrix and human cord blood (CB) cells to simulate the marrow microenvironment and expand cord progenitors. Nonwoven polyethylene terephthalate (PET) fabric with defined microstructure was used as the 3-D scaffold and the PET surface was modified by hydrolysis to improve cell adhesion. Different cell organizations were formed in a 3-D matrix in a developmental manner, from individual cells and cells bridging between fibers to large cell aggregates. Both stromal and hematopoietic cells were distributed spatially within the scaffold. Compared to two-dimensional (2-D) CD34(+) cell culture, 3-D culture produced 30-100% higher total cells and progenitors without exogenous cytokines. With thrombopoietin and flt-3/flk-2 ligand, it supported two- to three-fold higher total cell number (62.1- vs. 24.6-fold), CD34(+) cell number (6.8- vs. 2.8-fold) and colony-forming unit (CFU) number for 7-9 weeks (n = 6), indicating a hematopoiesis pathway that promoted progenitor production. Culture in 3-D nonwoven matrices enhanced cell-cell and cell-matrix interactions and allowed 3-D distribution of stromal and hematopoietic cells. The formation of cell aggregates and higher progenitor content indicated that the spatial microenvironment in 3-D culture played an important role in promoting hematopoiesis. This 3-D culture system can be used as an in vitro model to study stem cell or progenitor behavior, and to achieve sustained progenitor expansion.

Precancerous Stem Cells Have the Potential for both Benign and Malignant Differentiation

Cancer stem cells (CSCs) have been identified in hematopoietic and solid tumors. However, their precursors—namely, precancerous stem cells (pCSCs) —have not been characterized. Here we experimentally define the pCSCs that have the potential for both benign and malignant differentiation, depending on environmental cues. While clonal pCSCs can develop into various types of tissue cells in immunocompetent mice without developing into cancer, they often develop, however, into leukemic or solid cancers composed of various types of cancer cells in immunodeficient mice. The progress of the pCSCs to cancers is associated with the up-regulation of c-kit and Sca-1, as well as with lineage markers. Mechanistically, the pCSCs are regulated by the PIWI/AGO family gene called piwil2. Our results provide clear evidence that a single clone of pCSCs has the potential for both benign and malignant differentiation, depending on the environmental cues. We anticipate pCSCs to be a novel target for the early detection, prevention, and therapy of cancers.

Prolonged continuous in vitro human platelet production using three-dimensional scaffolds

OBJECTIVE Methods producing human platelets using growth on plastic, on feeder layers, or in suspension have been described. We hypothesized that growth of hematopoietic progenitors in a three-dimensional (3D) scaffold would enhance platelet production sans feeder layer. MATERIALS AND METHODS We grew CD34 positively selected human cord blood cells in surgical-grade woven polyester fabric or purpose-built hydrogel scaffolds using a cocktail of cytokines. RESULTS We found production of functional platelets over 10 days with two-dimensional (2D), 24 days with 3D scaffolds in wells, and more than 32 days in a single-pass 3D perfusion bioreactor system. Platelet numbers produced daily were higher in 3D than 2D, and much higher in the 3D perfusion bioreactor system. Platelet output increased in hydrogel scaffolds coated with thrombopoietin and/or fibronectin, although this effect was largely obviated with markedly increased production caused by changes in added cytokines. In response to thrombin, the platelets produced aggregated and displayed increased surface CD62 and CD63. CONCLUSION Use of 3D scaffolds, especially in a bioreactor-maintained milieu, may allow construction of devices for clinical platelet production without cellular feeder layers.

Diaspirin-crosslinked hemoglobin reduces blood transfusion in noncardiac surgery: a multicenter, randomized, controlled, double-blinded trial.

In this randomized, prospective, double-blinded clinical trial, we sought to investigate whether diaspirin-crosslinked hemoglobin (DCLHb) can reduce the perioperative use of allogeneic blood transfusion. One-hundred-eighty-one elective surgical patients were enrolled at 19 clinical sites from 1996 to 1998. Selection criteria included anticipated transfusion of 2–4 blood units, aortic repair, and major joint or abdomino-pelvic surgery. Once a decision to transfuse had been made, patients received initially up to 3 250-mL infusions of 10% DCLHb (n = 92) or 3 U of packed red blood cells (PRBCs) (n = 89). DCLHb was infused during a 36-h perioperative window. On the day of surgery, 58 of 92 (64%; confidence interval [CI], 54%–74%) DCLHb-treated patients received no allogeneic PRBC transfusions. On Day 1, this number was 44 of 92 (48%; CI, 37%–58%) and decreased further until Day 7, when it was 21 of 92 (23%; CI, 15%–33%). During the 7-day period, 2 (1–4) units of PRBC per patient were used in the DCLHb group compared with 3 (2–4) units in the control patients (P = 0.002; medians and 25th and 75th percentiles). Mortality (4% and 3%, respectively) and incidence of suffering at least one serious adverse event (21% and 15%, respectively) were similar in DCLHb and PRBC groups. The incidence of jaundice, urinary side effects, and pancreatitis were more frequent in DCLHb patients. The study was terminated early because of safety concerns. Whereas the side-effect profile of modified hemoglobin solutions needs to be improved, our data show that hemoglobin solutions can be effective at reducing exposure to allogeneic blood for elective surgery.

Separation of a breast cancer cell line from human blood using a quadrupole magnetic flow sorter

We have developed a quadrupole magnetic flow sorter (QMS) to facilitate high‐throughput binary cell separation. Optimized QMS operation requires the adjustment of three flow parameters based on the immunomagnetic characteristics of the target cell sample. To overcome the inefficiency of semiempirical operation/optimization of QMS flow parameters, a theoretical model of the QMS sorting process was developed. Application of this model requires measurement of the magnetophoretic mobility distribution of the cell sample by the cell tracking velocimetry (CTV) technique developed in our laboratory. In this work, the theoretical model was experimentally tested using breast carcinoma cells (HCC1954) overexpressing the HER‐2/neu gene, and peripheral blood leukocytes (PBLs). The magnetophoretic mobility distribution of immunomagnetically labeled HCC1954 cells was measured using the CTV technique, and then theoretical predictions of sorting recoveries were calculated. Mean magnetophoretic mobilities of (1−3) × 10−4 mm3/(T A s) were obtained depending on the labeling conditions. Labeled HCC1954 cells were mixed with unlabeled PBLs to form a “spiked” sample to be separated by the QMS. Fractional recoveries of cells for different flow parameters were examined and compared with theoretical predictions. Experimental results showed that the theoretical model accurately predicted fractional recoveries of HCC1954 cells. High‐throughput (3.29 × 105 cells/s) separations with high recovery (0.89) of HCC1954 cells were achieved.

Culturing and differentiation of murine embryonic stem cells in a three-dimensional fibrous matrix

Embryonic stem (ES) cells have indefinite self-renewal ability and pluripotency, and can provide a novel cell source for tissue engineering applications. In this study, a murine CCE ES cell line was used to derive hematopoietic cells in a 3-D fibrous matrix. The 3-D matrix was found to maintain the phenotypes of undifferentiated ES cells as indicated by alkaline phosphatase (ALP) activity and stage specific embryonic antigen-1 (SSEA-1) expression. In hematopoietic differentiation, cells from 3-D culture exhibited similar cell cycle distribution and SSEA-1 expression to those in the initial cell population. The Oct-4 expression was significantly down-regulated, which indicated the occurrence of differentiation, although the level was slightly higher than that in Petri dish culture. The expression of c-kit, cell surface marker for hematopoietic progenitor, was higher in the 3-D culture, suggesting a better-directed hematopoietic differentiation. Cells in the 3-D matrix tended to form large aggregates associated with fibers. For large-scale processes, a perfusion bioreactor can be used for both maintenance and differentiation cultures. As compared to the static culture, a higher growth rate and final cell density were resulted from the perfusion bioreactor due to better control of the reactor environment. At the same time, the differentiation capacity of ES cells was preserved in the perfusion culture. The ES cell culture in the fibrous matrix thus can be used as a 3-D model system to study effects of extracellular environment and associated physico-chemical parameters on ES cell maintenance and differentiation.

Manipulation of Oxygenation and Flow-Induced Shear Stress Can Increase the In Vitro Yield of Platelets from Cord Blood

A method to produce clinically useful platelets in vitro would help overcome the frequent shortages, donor deferrals, disease transmission, and alloimmunization with volunteer donor-derived platelets. Using CD34 positively selected cord blood cells, we investigated ways to increase platelet quality and yield in a three-dimensional modular perfusion bioreactor system. We found a two- to threefold increase in platelet numbers produced only when the early phases of the culture process were carried out at 5% oxygen, versus when 20% oxygen was used throughout the culture period (p<0.05), and much more than when 5% oxygen was used throughout. When the medium was routed through the cell-scaffold construct, versus when it flowed under and over the construct, or just intermittent feeding was used, the number of platelets increased two- to threefold (p<0.05), and enhanced collagen-induced aggregation. The 5% oxygen early in the culture process mimics the marrow adjacent to the bone where early progenitors proliferate. Flow through the cell-scaffold construct creates shear forces that mimic the flow in central venous sinuses of the marrow and enhances platelet production from proplatelets. The use of altered oxygen levels and cross flow enhanced platelet numbers and quality, and will contribute to eventual in vitro platelet production for clinical use.

Evaluation of Eluents from Separations of CD34+ Cells from Human Cord Blood Using a Commerical, Immunomagnetic Cell Separation System

Human CD34+ cells from cord blood were separated in a two‐step process using a commercial, immunomagnetic cell retention system. The performance of the system was evaluated by analyzing a number of eluents from the separations with a number of analytical techniques. In addition to cell counts and flow cytometry analysis, a new experimental technique that is undergoing development, cell tracking velocimetry (CTV), was used. CTV measures the degree to which a cell is immunomagnetically labeled, known as the magnetophoretic mobility, of a population of cells on a cell‐by‐cell basis and presents the results in the form of a histogram similar to flow cytometry data. The average recovery and purity of CD34+ cells from 10 separations was 52% and 60%, respectively. CTV analysis indicated that the mean magnetophoretic mobility of the positively enriched CD34 cells was 9.64 × 10−5 mm3/T‐A‐s, while the mean mobility from negative eluents was −2.02 × 10−6 mm3/T‐A‐s, very similar to the mobility of unlabeled cells. Within the positive eluents, the range of magnetophoretic mobility was approximately 50‐fold, representing a plausible 50‐fold range in surface CD34 antigen expression. CTV analysis also indicated that in some separations, positive cells were not retained by the immunomagnetic cell retention system. Finally, preliminary studies indicate that monocytes might be a primary cause in the lower purities and recoveries seen in this study. It is suggested that the monocytes phagocytose the magnetic nanobeads and become sufficiently magnetized to be retained within the Miltenyi column, reducing the purity of the positive eluent.