Kjell Bertheussen

Echinoid phagocytes in vitro

Abstract A method is described for obtaining pure monolayers of phagocytes from the sea urchin Strongylocentrotus droebachiensis in vitro. The coelomic fluid contains four types of cells. About 67% of the cells are phagocytes, the rest is comprised of the red and white morula cells and the vibratile cells. The different cell types could be separated by centrifugation on a discontinuous gradient of sodium metrizoate. Release of granula from the vibratile cells was found to be responsible for rapid and extensive clotting of the coelomic fluid immediately after its removal from the animal. Clotting was prevented by adding a mixture of 50 mM mercaptoethanol, 3 mM caffeine and 2 mM TAME ( p -tosyl- l -arginine methyl ester) to the coelomic fluid. The phagocytes were isolated from other cell types by their attachment to glass, and were grown at 10 °C in a simple peptone-sea water medium. The phagocytes are very motile cells and spread rapidly on glass, accompanied by a complete change of their morphology to flattened cells with peripheral ruffling. After few hours in vitro the cells fuse to form monolayer-syncytia, and later still cell clusters and free floating balls of cells are formed. During a culture period of 10 days there was no change in the DNA content per culture, while a small increase in protein was found.

Complement-like activity in sea urchin coelomic fluid

A complement-like activity in echinoid coelomic cell-free fluid is described. The activity is detected by the lytic action on rabbit erythrocytes (RRBC), and by the opsonic effect on echinoid coelomic phagocytes and mouse peritoneal macrophages. This activity is very heat-labile, being completely destroyed at 37 degrees C 1/2 hr, and is inhibited by Ca2+ concentrations below 10 mM and by low pH. Lysis was complete within 3-4 min, and the titer (10(7) RRBC/ml) was 20-60 between animals. Various substances known to inhibit human complement also inhibited the lytic and opsonic activities in echinoid fluid. RRBC opsonized with echinoid fluid were attached to mouse macrophages without being internalized, an effect which resemble complement opsonization. It is concluded that an activity is present in echinoid coelomic fluid, which strongly resembles mammalian complement activated via the alternative pathway. A lectin-like activity with specificity for D-fucose was detected by the agglutination of RRBC (titer 300-600). Hemagglutination was inhibited by sugars which did not inhibit the lytic and opsonic process. On the other hand, hemagglutination was resistant to various physio-chemical treatments which led to inactivation of the complement-like system; thus these two activities seem to be unrelated. The lectin-like activity did not mediate any opsonic effect.

Receptors for Complement on Echinoid Phagocytes. I. The Opsonic Effect of Vertebrate Sera on Echinoid Phagocytosis

The ingestion of sheep erythrocytes (SRBC) by echinoid phagocytes was greatly increased after treatment of SRBC with sera from mouse, human and fish. The opsonic principle in the sera was heat labile (56 degrees C 1/2 hr). Opsonization with mammalian sera was studied more extensively. It was dependent on pre-sensitization of SRBC with specific antibody (IgM), required Ca2+, and was inhibited by low temperature (4 degrees C). The finding that serum depleted of C 3 only opsonized very weakly strongly indicates that the opsonic principles is the complement cascade C 1-4-2-3 activated via the classical pathway, and that the opsonic effect largely coincides with the coating of SRBC with C 3b. A role of C 5 or later components was excluded, since the mouse AKR serum was C 5 deficient, and the human serum was normally treated with zymosan. When variations known to impair the opsonic function of C 3 was introduced in the opsonization procedure, a parallel inhibition was recorded on attachment of SRBC to mouse peritoneal macrophages and on ingestion of SRBC by echinoid cells. Thus C 3 receptors are probably present on echinoid phagocytes.

The cytotoxic reaction in allogeneic mixtures of echinoid phagocytes

Echinoid phagocytes recognize allogeneic and xenogeneic echinoid cells in vitro. This is demonstrated by the occurrence of a cytotoxic reaction after 20 h when phagocytes from different animals are cultured together. Within the species about 70% of different cell combinations gave cytotoxicity, while the frequency of reactivity increased to more than 90% when cells from different species were mixed. Target cells detach and disintegrate rapidly into small fragments. Most target cells seem to be killed between 20 and 36 h. Formation of syncytia is to some extent inhibited prior to the cytotoxic reaction. No cell division occurs during normal cell culture or during the cytotoxic events in vitro. The reaction is contact-dependent, bilateral and is often very strong. In most cases cytotoxicity levels of 20–40% were noted according to 51Cr-release assays. This together with the morphological datas implies that there is a very high proportion of reactive cells in any given allogeneic or xenogeneic cell combination. The mixed phagocyte reaction was not affected by presence of other coelomic cell types. The invertebrate phagocytic cell is probably also the effector cell during graft rejection and may represent the common ancestor of the vertebrate macrophage and lymphocyte.

Growth of cells in a new defined protein-free medium

The development of a new stable synthetic serum replacement (SSR) is described, which allows the cultivation of mammalian cells in a defined, protein-free medium containing only dialyzable components. With a low concentration of insulin (RPMI-SR2 medium), growth rates of the transformed cell lines L929, HELA S3, and the hybridoma 1E6 were comparable to growth rates obtained with a serum-containing medium. The same medium also supported long-term cultivation of non-dividing mouse macrophages. The main principle of SSR is a metal ion buffer containing a balanced mixture of iron and trace metals. Stability against precipitation of important metals is achieved by the combined use of EDTA and citric acid as chelating agents. Efficient iron supply is mediated through the inclusion of the compound Aurintricarboxylic acid as a synthetic replacement for transferrin. SSR also contains a growth-promoting surfactant, Pluronic F68. Thus SSR provides a general foundation for growth and differentiation normally provided by serum.Limitations of other serum-free medium designs are discussed here: 1) the inability of transferrin to chelate all metals in the medium; and 2) the use of inorganic iron salts or iron citrate as an iron supplement leads to rapid precipitation of iron hydroxide in the medium. Both these problems are solved in the design of SSR.

Optimization and simplification of culture conditions in human in vitro fertilization (IVF) and preembryo replacement by serum-free media

The results of 220 consecutive IVF treatments are presented, comparing the use of culture media supplemented with either patient serum (Group 1;n=110), or Medi-Cult SSR 2 synthetic serum replacement with pyruvate, and human serum albumin (HSA) (GEA BioTech, Hvidovre, Denmark) (Group 2;n=110). In both groups the Medi-Cult Hybritest was used for routine quality testing. A significantly (P<0.05) increased rate of deliveries/ongoing pregnancies was observed with the Group 2 medium. However, no significant differences in fertilization rate, cleavage rate, or implantation rate were observed. It is concluded that the serum-free culture medium described and the testing for absence of cytotoxicity in a sensitive bioassay (Hybritest) have yielded culture conditions capable of sustaining the development in vitro of human preembryos without impairing the fertilization process or the implantation rate, ultimately resulting in a significantly increased rate of deliverieslongoing pregnancies and an apparently decreased abortion rate. The porential harmful effects of serum and the need for blood sampling and preparation further increase the advantages of replacing serum with the synthetic serum replacement SSR 2 in an IVF program.

Endocytosis by echinoid phagocytes in vitro I. Recognition of foreign matter

Endocytosis, with emphasis on phagocytic recognition was examined in the sea urchin Strongylocentrotus droebachiensis. Most cells (about 67%) in the coelomic fluid were phagocytes. There was also a linear uptake of colloidal gold with time in the cultured cells, presumably caused by pinocytosis. No opsonizing activity was found in the cell-free coelomic fluid, and the parallel phagocytic avidity in vivo and in vitro of different particles also indicates that humoral factors were absent or not essential for recognition of foreign surfaces by phagocytes. Untreated erythrocytes (RBC) were taken up at a low rate, while RBC treated with aldehyde, Fe2+, tannin, Con A, or IgM + mouse C5-deficient complement were phagocytosed efficiently. Phagocytosis was reduced when RBC were covered with specific antibodies. Thus, the echinoid phagocyte lacks an Fc receptor, but may have a C3b receptor on its membrane. Other particles with high phagocytic avidities were carbon, Sephadex, latex and Escherichia coli. Thus the cell membranes of phylogenetically distant phagocytes may exhibit common structural features responsible for foreign surface recognition.

Differential pH in embryo culture

OBJECTIVE To determine the optimum pH in sequential media for embryo culture around the fertilization-zygote stage and cleavage stage, with use of a mouse embryo assay. DESIGN Experimental laboratory study. SETTING University Hospital and University Research Unit. ANIMAL(S) F1 hybrids between CD1 female and BDF male mice. INTERVENTION(S) Fertilized, one-cell mouse embryos were cultured 5 days in test media where pH was changed at defined time intervals. MAIN OUTCOME MEASURE(S) Percentage of good-quality embryos, defined by strict morphology. RESULT(S) A significantly improved development was observed when pH was as high as 7.30 before the pronuclear stage and lowered to pH 7.15 during the cleavage period. CONCLUSION(S) Good embryo development is consistent with two different pH values in sequential culture media. This could have important implications for embryo culture in human IVF.

Endocytosis by Echinoid Phagocytes in Vitro II. Mchanisms of Endocytosis

Abstract The mechanism of endocytosis was examined in the sea urchin Strongylocentrotus droebachiensis . Phagocytosis proceeds most efficiently at 5-15 °C, and when the cells are not so spread out on the substrate. Iodoacetate inhibited both phagocytosis and pinocytosis, while only pinocytosis was sensitive to cyanide, suggesting different metabolic requirements for the two processes. Cytochalasin B exhibited a differential effect on phagocytosis, depending on the nature of the particles surface. A possible mechanism involving interference of cytochalasin B with the recognition stage of phagocytosis is discussed. Colchicine did not affect phagocytosis.

New media for culture to blastocyst

OBJECTIVE To develop sequential media for extended culture of embryos in human IVF, with use of a mouse embryo assay. The BlastAssist system was developed. Blastocyst culture has been our routine method since 1994, using earlier media IVF and M3 in sequence. DESIGN Experimental laboratory study. SETTING University Hospital and university research unit. ANIMAL(S) F1 hybrids between CD1 female and BDF male mice. INTERVENTION(S) Two-cell mouse embryos were cultured 4 days in test media; 1 day in phase 1 medium followed by 3 days in phase 2 medium. MAIN OUTCOME MEASURE(S) Percentage of expanded and hatched blastocysts. RESULT(S) Compared with a two-cell block in the old culture system, more than 80% went to the blastocyst stage in the new media. The new medium system was based on a heavily modified low-glucose Earles balanced salt solution. Additions were pyruvate, lactate, amino acids, human serum albumin, and Synthetic Serum Replacement. In contrast to regular tissue culture cells, embryos required an increased concentration of EDTA. The final media produced a blastocyst frequency of 94% and a day 4 hatching frequency of 71%. CONCLUSION(S) With the high blastocyst frequency demonstrated here, the combination of blastocyst culture and single ET should be an effective means of treatment in patients and could help to eliminate multiple gestations. The BlastAssist system is commercially available.