Ex vivo culture systems for hematopoietic cells.

During the past few years, hematopoietic cell culture technologies for transplantation therapies have progressed significantly on several fronts. Advances include the discoveries of the growth factors thrombopoietin and Flt-3 ligand, the development of a variety of bioreactor systems, and results from preliminary clinical trials that demonstrate the efficacy of ex vivo expanded hematopoietic cells for transplantation therapy.

Oxygen tension modulates the expression of cytokine receptors, transcription factors, and lineage-specific markers in cultured human megakaryocytes.

OBJECTIVE: We have recently reported that 20% O2 significantly enhances total megakaryocyte (Mk) number, polyploidy, and proplatelet formation compared to 5% O2 in culture. In order to further elucidate the regulatory role of pO2 on megakaryocytopoiesis, we conducted a kinetic study of the expression of surface markers CD41a and CD42a; receptors for thrombopoietin (TPO), interleukin-3 (IL-3), and Flt3-ligand; the glutamate receptor of the N-methyl-D-aspartate subtype 1 (NMDAR1); and transcription factors GATA-1, NF-E2, and E2F-1. MATERIALS AND METHODS: Mks were generated from mobilized peripheral blood (PB) CD34+ cells from normal donors in serum-free medium with TPO, IL-3, and Flt3-ligand at 20% and 5% O2. Quantitative assessment of Mk surface receptors and nuclear transcription factors was performed using multiparameter flow cytometry. mRNA levels of the nuclear transcription factors GATA-1 and NF-E2 were evaluated using RT-PCR. RESULTS: The proportions of cells expressing the early Mk marker CD41a and the late Mk marker CD42a at day 15 were 4 and 5 times higher, respectively, at 20% O2. CD41a and CD42a protein levels per cell were also higher at 20% O2. After day 5, c-Mpl (TPO receptor) generally followed similar kinetics as CD41a. The proportion of IL-3 receptor (IL-3R)++ Mks at day 5 was 1.5 times higher at 5% O2. The NMDAR1 protein previously known to be expressed by neuronal cells has recently been identified in Mks. NMDAR1 and the transcription factors were studied on days 6, 9, and 11. NMDAR1 was expressed at a 1.5- to 1.8-fold higher level at 5% O2. Twenty percent O2 supported higher expression of the Mk-early and -late-maturation-specific transcription factors GATA-1 (1.2- to 2.2-fold higher) and NF-E2 (1.1- to 2.8-fold higher). This was consistent with RT-PCR data indicating the presence of higher levels of GATA-1 and NF-E2 mRNA at 20% O2. E2F-1, a ubiquitously expressed cell cycle transcription factor, was expressed at a 1.5-fold higher level at 20% O2 on day 6, but this difference did not persist by day 9. CONCLUSION: These findings demonstrate that cytokine receptors c-Mpl and IL-3R, and Mk differentiation-specific surface receptors CD41a, CD42a, and NMDAR1, are significantly modulated by pO2, and suggest that one of the mechanisms of enhanced maturation at 20% O2 may involve regulation of transcription factors GATA-1 and NF-E2.

Physiologically significant effects of pH and oxygen tension on granulopoiesis.

OBJECTIVE: Granulocyte differentiation in the bone marrow (BM) takes place in regions with lower pH and O(2) tension (pO(2)) than those in the BM sinuses. This suggests that granulopoiesis will be enhanced at subvascular pH and pO(2). MATERIALS AND METHODS: The effects of pH AND pO2 on granulocyte proliferation, differentiation, and granulocyte colony-stimulating factor receptor (G-CSFR) expression were evaluated using mobilized peripheral blood CD34(+) cells directed down the granulocytic pathway with stem cell factor, interleukin 3, interleukin 6, and G-CSF. RESULTS: Cell expansion was enhanced at subvascular pH, with twice as many total cells and CD15(bright)/CD11b(+) late neutrophil precursors (myelocytes, metamyelocytes, bands) produced at pH 7.07 to 7.21 as was produced at pH 7.38. Low pH accelerated the rate of differentiation concomitant with this increase in proliferation. Also, total, CD15(bright)/CD11b(-) (promyelocytes, early myelocytes), and CD15(bright)/CD11b(+) cell expansion was enhanced at lower pO(2), with twice as many of each cell type produced at 5% O(2) as at 20% O(2). The effects of low pH and low pO(2) were additive, such that generation of total, CD15(bright)/CD11b(-), and CD15(bright)/CD11b(+) cells was 3.5-, 2.4-, and 4.0-fold greater at pH 7.21 and 5% O(2) than at the standard hematopoietic culture conditions of pH 7.38 and 20% O(2). Low pH resulted in faster upregulation of G-CSFR surface expression, whereas pO(2) had no effect on G-CSFR expression. CONCLUSION: These data provide compelling evidence that pH and pO(2) gradients within the BM play significant roles in regulating hematopoiesis. More rapid granulocytic cell proliferation and differentiation at low pH may be explained in part by more rapid G-CSFR expression. The ability to alter cell development by manipulating pH and pO(2) has important implications for optimizing ex vivo production of neutrophil precursors.

Oxygen tension alters the effects of cytokines on the megakaryocyte, erythrocyte, and granulocyte lineages.

Many investigators believe that cytokines have exclusive control over proliferation and differentiation in stroma-free hematopoietic cultures. Although cytokines are indeed necessary, other culture parameters such as oxygen (O2) tension can greatly influence both hematopoietic cell proliferation and differentiation. We investigated the effects of cytokine combinations and O2 tension on the expansion of megakaryocyte (size and number of CD41a+ cells and number of colony-forming units-megakaryocyte [CFU-Mk]), granulocyte (CD15+ cells and CFU-granulocyte monocyte [CFU-GM]), and erythrocyte (hemoglobin+ cells and burst-forming units-erythrocyte [BFU-E]) lineages. Peripheral blood CD34+ cells were cultured in serum-free medium with interleukin (IL)-3, stem cell factor (SCF), and various combinations of thrombopoietin (TPO), erythropoietin (EPO), and/or granulocyte-colony stimulating factor (G-CSF). The effects of TPO, EPO, and G-CSF on Mks, erythrocytes, and granulocytes, respectively, were dependent on the O2 tension. Thrombopoietin-containing cultures under a gas phase of 20%) O2 tension produced 1.4- to 2.2-fold more Mks than those under 5% O2. The increase in Mk size with TPO was also much greater under 20% O2. Similarly, 2.1- to 2.4-fold more hemoglobin-containing cells were produced in EPO-containing cultures under 20% vs. 5% O2. In contrast, approximately twice as many CD15+ cells were produced in G-CSF-containing cultures under 5% vs. 20%) O2. The numbers of CFU-Mk in TPO-containing and CFU-GM in G-CSF-containing cultures were larger under 5% O2. Although the O2 tension had no effect on BFU-E production in EPO-containing cultures, BFU-E production under 5% O2 in cultures without EPO was equal to that in EPO-containing cultures. Our data also suggest that TPO, EPO, and G-CSF elicit stimulatory cross-lineage effects in the presence of IL-3 and SCF, and that these effects, too, are often dependent on O2 tension.

Reduced oxygen tension increases hematopoiesis in long-term culture of human stem and progenitor cells from cord blood and bone marrow.

Growth of hematopoietic stem and progenitor cells found in the mononuclear cell (MNC) fraction of human cord blood and bone marrow was evaluated under atmospheres containing reduced (5%) and normal (20%) oxygen tension. Cord blood MNC were grown in suspension and on preestablished irradiated bone marrow stromal layers, whereas bone marrow MNC were used to initiate one-step long-term bone marrow cultures (LTBMC). Reduced oxygen tension resulted in a substantial increase in both the number and frequency of colony-forming cells observed in all three types of long-term hematopoietic cultures (LTHC) studied. At various time points under low oxygen, progenitor cell numbers were as much as 12-fold, 3-fold, and 4-fold higher for granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E), and granulocyte erythrocyte macrophage megakaryocyte colony-forming units (CFU-GEMM), respectively. In addition to these numerical increases, progenitor cells were maintained for 1-2 weeks longer under low oxygen conditions. Reduced oxygen tension also increased total cell numbers by as much as fivefold in cord blood suspension cultures, but this effect on total cell numbers was less pronounced in cultures containing a stromal layer. The rate of irradiated stromal layer degeneration, as judged by cell counts and microscopic examination, was reduced under low oxygen. Finally, the beneficial effect of reduced oxygen was comparable to the effect of an irradiated stromal layer for maintaining cord blood progenitor cells in LTHC. These results indicate that low oxygen, which better approximates the in vivo environment, enhances the growth and maintenance of both stromal and progenitor cells for a longer period of time in vitro.

Dynamic model of ex vivo granulocytic kinetics to examine the effects of oxygen tension, pH, and interleukin-3.

OBJECTIVE: Evaluating kinetics in hematopoietic cultures is complicated by the distribution of cells over various stages of differentiation and by the presence of cells from different lineages. Thus, an observed response is an integral response from distributed cell populations. Growth factors and other parameters can greatly affect the lineage and maturation stage of the culture outcome. To resolve the kinetics and more clearly define the differential effects of O(2) tension (pO(2)), pH, and interleukin-3 (IL-3) on granulopoiesis, a mathematical model-based approach was undertaken. MATERIALS AND METHODS: Granulocytic differentiation is described within a continuous, deterministic framework in which cells develop from primitive granulocytic progenitors to mature neutrophils. The model predicts two distributed populations-quiescent and cycling cells-by incorporating rates of growth, death, differentiation, and transition between quiescence and active cycling. The response of these four model processes to changes in the culture environment was examined. RESULTS: Model simulations of experimental data revealed the following: 1) pO(2) effects are exerted only on the growth rate but not maturation times. 2) pH effects between pH 7.25 and 7.4 on growth and differentiation are coupled; however, with increasing pH values, especially at pH 7. 6, the death rate for cells in the early stages of differentiation becomes increasingly significant. 3) The absence of IL-3 increases the death rate for primitive cells only minimally but markedly enhances the rate of differentiation through the myeloblast window in the differentiation pathway. The combined effects of these environmental factors can be predicted based on changes in the model parameters derived from the individual effects. CONCLUSIONS: Experimental data combined with mathematical modeling can elucidate the mechanisms underlying the regulation of granulopoiesis by pO(2), pH, and IL-3. The model also can be readily adapted to evaluate the effects of other culture conditions. The increased understanding of experimental results gained with this approach can be used to modify culture conditions to optimize ex vivo production of neutrophil precursors.

Media additives for protecting freely suspended animal cells against agitation and aeration damage.

Over the past 30 years, several different additives have been used to protect freely suspended animal cells in culture from agitation and aeration damage. These include pluronic polyols, various derivatized celluloses and starches, protein mixtures, polyvinyl-pyrrolidones, dextrans, and, more recently, polyethylene glycol (PEG) and polyvinyl alcohol. The protective mechanisms of these additives are not yet fully understood, but recent studies suggest both fluid-mechanical and biological mechanisms of protection.

Fluid-mechanical damage of animal cells in bioreactors.

The fluid-mechanical and some biological aspects of damage to animal cells in bioreactors due to agitation and/or aeration are attracting renewed attention. In microcarrier bioreactors, cell damage is due to forces generated by the interaction of microcarrier beads with each other and also with small turbulent eddies. For freely suspended cells grown in mixed bioreactors, cell damage is most frequently due to bubble breakup or fast-draining liquid films around rearranging gas-liquid interfaces.

Hematopoietic cell culture therapies (Part I): Cell culture considerations.

Hematopoietic cell culture, or ex vivo expansion of hematopoietic cells, is a rapidly growing area of tissue engineering with many potential applications in bone-marrow transplantation, gene therapy and the production of blood products. Hematopoietic cultures are considerably more complex than established animal cell culture technologies owing to the presence of many cell types at various differentiation stages, and stringent medium and growth factor requirements. Culture parameters, such as pH, oxygen tension, seeding density and feeding schedules, significantly affect the proliferation and differentiation of hematopoietic cells. A number of bioreactor systems are currently under development.

Hematopoietic cell culture therapies (Part II): Clinical aspects and applications.

High-dose chemotherapy, followed by hematopoietic stem cell transplantation, holds significant promise for increasing the probability of long-term remission and possibly cure in a variety of cancers. Hematopoietic cell culture, or ex vivo expansion of hematopoietic cells, may play a significant role in reducing the danger and expense associated with the transplantation procedure. Phase I clinical trials have shown that ex vivo expanded cells have no significant toxicities, and some benefits. Ex vivo expansion of hematopoietic cells is likely to find other applications in gene therapy, tumor purging, production of dendritic cells for immunotherapy and the production of mature blood cells for transfusion therapies.

Sequence and arrangement of two genes of the butyrate-synthesis pathway of Clostridium acetobutylicum ATCC 824.

The genes encoding both Clostridium acetobutylicum ATCC 824 butyrate synthesis pathway enzymes, phosphotransbutyrylase (ptb) and butyrate kinase (buk), were sequenced. The genes are immediately adjacent on the chromosome, with ptb preceding buk. A single transcription start point (tsp) was identified 57 bp upstream from the ptb start codon by primer extension analysis. The ptb and buk genes appear to form an operon. A putative Rho-independent terminator structure was identified 26 bp downstream from buk.

Cell aggregation in a Chinese hamster ovary cell microcarrier culture affects the expression rate and N-linked glycosylation of recombinant mouse placental lactogen-1.

A microcarrier culture of Chinese hamster ovary (CHO) cells, expressing the N-glycosylated recombinant protein mouse placental lactogen I (mPL-I), was found to form large cellular aggregates (400 to 600 microns in diameter). There was increased accumulation of lower molecular sized mPL-I glycoforms in cultures containing the large cellular aggregates at pH 7.3, but not at pH 7.6. Specific rates of mPL-I expression were found in the cultures with cellular aggregates at both pH values (7.3 and 7.6). These findings are interpreted in the light of our earlier studies that showed that extracellular pH and elevated ammonia concentrations affect both the glycosylation and the expression rates of mouse placental lactogen I.

Methods for cloning key primary metabolic enzymes and ancillary proteins associated with the acetone-butanol fermentation of Clostridium acetobutylicum.

The unavailability of genetically defined mutants for complementation has intensified the problems inherent in cloning genes from C. acetobutylicum. The uniqueness of some of the pathways of this organism coupled with the relative inefficiency of transformation of clostridia and few characterized mutants in these pathways have made cloning these genes by traditional complementation methods impractical. Oligonucleotide hybridization techniques have been shown to circumvent many problems involved in detecting protein expression. The ease of hybridization screening of plaques allows phage libraries to be examined more readily than is generally the case with colony screening techniques. Recombinant lambda phages also contain more DNA per insert than most plasmid vectors can maintain, thus further decreasing the amount of screening necessary. Cosmid libraries, offering even greater length of individual inserts, can be screened in a similar manner, although such screening incorporates the limitations of colony screening techniques. It is true that the technique hinges on the ability to obtain an amino acid sequence from which an oligonucleotide can be designed. In the past, the ability to obtain sequences was limited because the quantity and number of purified proteins were limited or the proteins were amino-terminally blocked. However, recent technological advances in this area, such as high-resolution gel separation techniques coupled with microsequencing, have opened the door to proteins previously inaccessible. Deformylation methods have been developed to deblock amino-terminally formylated proteins, and successful internal amino acid sequence analysis by in situ protease digestion has also been reported using only picomolar quantities of proteins separated by one- or two-dimensional gel electrophoresis. Protein and DNA sequence data banks have been significantly upgraded in the past few years. A proposed oligonucleotide sequence can be evaluated to determine what other possible sequences have similar homology; moreover, protein similarity comparisons between related species might possibly supplant the need for protein isolation if regions of highly conserved amino acid sequences are found. To our knowledge, this represents the first reported use of oligonucleotide probe hybridization screening technology as a strategy for cloning solvent pathway genes of C. acetobutylicum. Despite the deleterious effects on hybridization inherent in the high A + T content of C. acetobutylicum gene specific-directed oligonucleotides, the technique has been shown to function with few modifications to previously recorded systems.

Transport of substrates and metabolites and their effect on cell metabolism (in butyric-acid and methylotrophic fermentations).

The two components, delta pH and delta psi, of the membrane protonmotive force (delta p) effect and are affected by the transport of many substrates and metabolites. Because the integrity (or restoration) of the delta p requires the expenditure of metabolic energy, such transport processes affect the overall cell bioenergetics. However, the transport or high concentrations of certain substrates and metabolites can have more serious effects on cell metabolism because they partially or completely abolish either or both the delta pH and delta psi. If the cells cannot eventually restore the collapsed component(s) of the delta p, complete growth inhibition and cell death become inevitable. In the butanol/acetone fermentation of Clostridium acetobutylicum, the transport and the presence of key metabolites (acetic and butyric acids, and butanol) have serious and some necessary effects on the delta p. Acetic and butyric acids act as uncouplers of the delta pH, thereby reducing the internal pH. Using other acid uncouplers (such as acetoacetate, which is metabolized by the cells, or FCCP, which is not metabolized by the cells), we found that a lower pHo combined with the metabolic-energy drain of the uncoupling effect and high internal acid concentrations are implicated in the mechanism(s) of solventogenesis. Thus, the production or presence (or both) of the two acids (acetic and butyric) is beneficial to the initiation of solvent production. The transport mechanisms of CH3OH, CH2O, and HCOOH in obligate CH3OH utilizers (methylotrophs) were also discussed in detail. We showed that CH3OH is actively transported by the cells at the expense of metabolic energy and that its transport significantly affects the dynamics of continuous bioreactors. The accumulation of CH2O was found to be driven by the membrane delta p. Finally, formate was accumulated by the delta pH according to the general transport mechanism of short-chain fatty acids. The inhibition of growth by formate was explained by its uncoupling effect on the cells. Growth inhibition by CH3OH appeared to be related to the severe reduction of the membrane delta pH and cell pHi by relatively low CH3OH concentrations.

Beneficial effects of reduced oxygen tension and perfusion in long-term hematopoietic cultures.

Growth of hematopoietic stem and progenitor cells found in the MNC fraction of human cord blood was evaluated under atmospheres containing reduced (5%) and normal (20%) oxygen tension. Reduced oxygen tension increased total cell numbers by as much as 5-fold in cord blood suspension cultures, but this effect was less pronounced in cultures containing an irradiated bone marrow stromal cell layer. However, reduced oxygen tension resulted in a substantial increase in both the number and frequency of colony-forming cells observed in both types of LTHC studied. Under low oxygen, CFU-C progenitor cell numbers were as much as 10-fold higher. Finally, reduced oxygen tension slowed the rate of irradiated stromal layer degeneration, as judged by cell counts and microscopic examination. These results indicate that low oxygen, which better approximates the in vivo environment, enhances the growth and maintenance of human stromal and progenitor cells in vitro. These low oxygen findings were then applied to a murine model LTHC perfusion system. In this system, irradiated 3T3 stromal layer integrity was improved under low oxygen and was substantially further improved with continuous medium perfusion. Cell counts and flow cytometry analysis indicated that the total cell production and the production of immature cells from murine bone marrow MNC on irradiated 3T3 cells were significantly enhanced under low oxygen with perfusion. After three weeks of culture, a 24-fold higher number of Thy1.2lo F4/80- MAC1- cells (indicative of murine stem and progenitor cells) was observed in the perfusion system as compared with static culture under ambient oxygen.

Vector construction, transformation, and gene amplification in Clostridium acetobutylicum ATCC 824.

In order to alter the primary metabolism of C. acetobutylicum, we have constructed E. coli- or B. subtilis-C. acetobutylicum shuttle vectors that could be used to deliver homologous fermentative genes into C. acetobutylicum ATCC 824. The plasmid copy number and plasmid stability in C. acetobutylicum for several of these plasmids were determined. We have also developed a protocol for the electrotransformation of C. acetobutylicum ATCC 824. Difficulty in the transformation of C. acetobutylicum ATCC 824 with vectors containing DNA from E. coli plasmids was found to be due to the existence of a restriction system in this strain. This type II restriction endonuclease, named Cac824I, recognizes the sequence 5'-GCNGC-3' and cuts ColE1 plasmids frequently. One of the vectors, pFNK1, possessing a variety of unique cloning sites was used in the amplification of one acid (PTB) and one solvent (AADC) formation gene. The corresponding enzyme activities were amplified in C. acetobutylicum as shown by enzyme assays and SDS-PAGE gels of cell extracts.

Cloning of an NADH-dependent butanol dehydrogenase gene from Clostridium acetobutylicum.

The acetone-butanol fermentation of C. acetobutylicum is characterized by the unique shift from acid to solvent production. The mechanism of the solventogenic switch involves the induction of several enzymes, including NADH-dependent butanol dehydrogenase (BDH) at the onset of solventogenesis. This enzyme is responsible for the final conversion of butyraldehyde to butanol, and is distinct from the NADPH-dependent alcohol dehydrogenase (ADH) also present in the organism. To characterize the genetic control of this gene, we have cloned and expressed it in E. coli. A lambda EMBL3 phage library of C. acetobutylicum DNA was screened via plaque hybridization using a [32P]-radiolabeled, 32-fold degenerate, 62-mer oligonucleotide probe. The probe was designed by reverse translation of the NH2-terminal amino acid sequence of purified BDH II. Southern blot experiments indicate that the phage insert was of clostridial origin and had no homology with the previously cloned NADPH-dependent ADH. Subcloning of DNA from purified positive plaques has localized the gene to a 3.5-kb EcoRI fragment from which the enzyme is well expressed. The sequence of the 25 NH2-terminal amino acids for the cloned enzyme purified from E. coli was determined and found to be identical to that for the clostridial NADH-dependent BDH II. Maxicell analysis of [35S]-radiolabeled plasmid-encoded proteins identified a species encoded by the clostridial insert with the expected Mr of 42 kD.

Determination of plasmid copy number and stability in Clostridium acetobutylicum ATCC 824.

The copy number and stability of several plasmid vectors in Clostridium acetobutylicum ATCC 824 were determined. The protocols were modified from the traditional ones to overcome the problems associated with unusual behavior of C. acetobutylicum cells on solid medium. The plasmid copy numbers of pSYL2, pFNK1, pFNK3, and pFNK5 in strain ATCC 824 were 14, 8, 6, and 6, respectively. pSYL2 and pFNK1 were segregationally stable, since the fractions of plasmid-carrying cells after 60 generations of growth without antibiotic (erythromycin) were 73% and 77%, respectively. Vector pFNK1 carrying fermentative genes was found to be rather unstable. The observed instability seemed to be due to the complex host-plasmid interactions by amplified expression of enzymes involved in the tightly regulated primary metabolism of C. acetobutylicum.

The protective effect of serum against hydrodynamic damage of hybridoma cells in agitated and surface-aerated bioreactors.

The effect of serum on the growth rate and metabolism of CRL-8018 hybridoma cells in an agitated, surface-aerated bioreactor was examined. In the employed well-controlled bioreactors at high agitation rates, hybridoma cells in medium containing 1% fetal bovine serum rapidly die in the presence of a vortex with accompanying gas-bubble entrainment, whereas non-agitated control cultures grow normally in medium containing 1% serum. Serum levels greater than 5% counteract the detrimental hydrodynamic effects due to agitation and bubble entrainment. The protective effect is present after short-term (less than 1 h) exposure to 10% serum concentration, suggesting a protection mechanism which is, at least in part, of a physical nature. The apparent cell yields on glucose, lactate, and glutamine decreased with decreasing growth rate due to low serum concentrations. The results are incorporated into a simple model in which the apparent growth rate is the sum of an invariable growth rate and a changing death rate.

Polyvinyl alcohol and polyethylene glycol as protectants against fluid-mechanical injury of freely-suspended animal cells (CRL 8018).

Two identical bioreactors run in parallel were used to examine the phenomenological characteristics of two additives, polyethylene glycol (PEG) and polyvinyl alcohol (PVA), used as protectants against fluid-mechanical cell damage. Cell-protecting ability was evaluated by comparing apparent cell growth rates of freely suspended CRL-8018 hybridoma cells cultured in serum-free medium under surface aerated conditions whereby cell damage is due to bubble entrainment and breakup. PEG of various molecular weights was used to determine whether the size of the polymer has significant effects on PEG's cell-protecting capabilities. All the PEG's with molecular weights larger than 1400 showed similar protective effects. The effect of PEG concentration was then evaluated and results showed that concentrations greater than 0.05% w/v did not significantly improve the cell-protecting properties. Direct comparisons made between the PVA, PEG, and pluronic F68 as cell protectants showed that PEG protected cells better than F68 and that PVA provided even better protection than PEG. The mechanism of protection, fluid-mechanical or biological in nature, was examined by growing the cells in additive from the beginning of the experiment (long-term exposure), or adding the additive after the cells had been agitated at rates detrimental to the cells (short-term exposure). In agreement with results reported previously on PEG and F68, fast-acting protection was seen. This implies a fluid-mechanical rather than a biological protection mechanism. In an attempt to correlate interfacial properties of the resulting media with shear protection, interfacial tension and viscosity measurements of all the media were made. On the basis of these measurements, we find no definitive correlations for evaluating these additives' cell-protecting capabilities.