Embedding Media for 1-2 Micron Sectioning. 3. Hydroxyethyl Methacrylate-Benzoyl Peroxide Activated with Pyridine

A hydroxyethyl methacrylate (HEMA) monomer medium containing 2-butoxyethanol as the plasticizer requites only one stock solution consisting of: 50 ml of 94 or 96% HEMA containing 200 ppm inhibitor (Rohm and Haas, Philadelphia) is mixed with 12 ml 2-butoxyethanol; 0.25 gm benzoyl peroxide is added and permitted to dissolve at 20-25 C. This mixture is activated by the addition of 0.8-1.0 ml of pyridine. Polymerization of the activated mixture is initated in 15-20 hr at 25 C and in 2-4 hr at 50 C; polymerization of the mixture is complete in 2-3 days and 3-6 hr, respectively. The activated monomer mixture is stable at temperatures below 18 C; hence infiltration of tissues may be extended to 7-10 days by keeping the mixture at 0 to -40 C.     

Hydroxyethyl Methacrylate Combined with Polyethylene Glycol 400 and Water; an Embedding Medium for Routine 1-2 Micron Sectioning

Pieces of tissue, with the largest dimension not exceeding 7 mm, are fixed and dehydrated by the procedures of choice. Two stock solutions: A, for infiltration; and B, the accelerator, are used in embedding. Formulas: A, 80 ml of glycol methacrylate (2-hydroxyethyl methacrylate—Rohm and Haas Co., Philadelphia, Pa.) is mixed well with 12 ml of polyethylene glycol (Carbowax) 400 and 8 ml of water; then 0.27 gm of benzoyl peroxide added, heated to dissolve the peroxide, and allowed to cool to room temperature. B, polyethylene 200 or 400, 15 parts, and N,N-dimethylaniline, 1 part, mixed thoroughly. Tissues are first infiltrated completely with solution A, then cast in a mixture consisting of 42 parts of A mixed with 1 part of B. Polymerization occurs in 45 min to 3 hr, depending on the temperature. In a water bath at 20 C, the time required was found to be about 3 hr; at 25 C, 1.5 hr; and at 30 C, 45 min. The plastic block can be trimmed easily, and sections 1-2 μ thick readily cut. Sections can be attached to slides by water flotation, without adhesive, and should be dried at room temperature. Staining with aqueous solutions of basic and acid dyes, without removing the embedding matrix, is sharp and brilliant. When staining of the matrix by basic dyes occurs, this background stain can be completely removed by differentiating in either 2-butoxyethanol, pure ethanol, or a mixture of the two. A number of histochemical reagents have been found compatible with this embedding procedure.     

Myelin impregnation: an improved Golgi-Cox modification.

Optic tecta of goldfish were coated with egg yolk and immersed for only one week in one of the following impregnation fluids: a) Solution A + B; A = 1 g K2Cr2O7 and 1 g HgCl2 boiled for 15 min in 85 ml distilled water and allowed to cool; B = 0.8 g K2Cr2O4 and 0.5 g KWO4 dissolved in 20 ml distilled water. b) Solution A + B two volumes diluted with boiled distilled water. c) Solution A + B four volumes diluted with boiled distilled water. Each tectum was immersed 6 hr in 100 ml distilled water containing 0.5 g LiOH and 15 g KNO3, washed 18 hr in 500 ml 0.2% acetic acid, dehydrated with ethanol, and embedded in low viscosity nitro cellulose. Sections were cut at 100 micron with a rotary microtome after clearing with cedarwood oil. Methods b) and c) have two advantages compared with method a), the original Golgi-Cox method. First, more cells are impregnated, especially in the layers extending 200-400 micron below the surface, and dendrites as well as unmyelinated axons are well impregnated. Second, myelin sheaths are impregnated and can be recognized by their peculiar chain-like appearance. The described Golgi-Cox modification offers an appropriate method to study the morphology of superficially located nervous tissue.     

The investigation of vinylcyclohexane dioxide as a polar dehydrant for the improved retention of lipids in the Spurr embedment

The use of vinylcyclohexane dioxide (VCD) as a polar dehydrant with subsequent embedment in Spurr was studied. The utilization of Epon 812 resin (E 812), hydroxyethyl methacrylate (HEM) and hydroxypropyl methacrylate (HPM) as polar dehydrants for Epon embedment were re-examined, and a polar Epon mix was introduced. The most effective dehydration sequence was: first 90%, then 95% VCD in water for 5 min. followed by two 20 min changes of 100% VCD. After 1 hr in equal quantities of VCD and Spurr mix, tissues were infiltrated with Spurr embedment (two 1 hr changes and overnight) and finally embedded in Spurr and polymerized at 60 degrees C for 16 hr. The most utilizable polar Epon mix was determined to be Epon 812 = 50 ml, NMA=42 ml, DMP-30=1-2 ml. It was somewhat brittle but cut well with both glass and diamond knives. All four polar dehydrants were found to retain lipids and carbohydrates equally well in thin section in striated and cardiac muscle, liver, kidney and brain from the rat. The E 812 was the only dehydrant that retained lung multilamellar bodies. The possible carcinogenic effects of VCD were considered and the probably metabolism and excretion of VCD were discussed.     

Myelin Impregnation: An Improved Golgi-Cox Modification

Optic tecta of goldfish were coated with egg yolk and immersed for only one week in one of the following impregnation fluids: a) Solution A + B; A = 1 g K₂Cr₂O₇ and 1 g HgCl₂ boiled for 15 min in 85 ml distilled water and allowed to cool; B = 0.8 g K₂Cr₂O₇ and 0.5 g KWO₄ dissolved in 20 ml distilled water, b) Solution A + B two volumes diluted with boiled distilled water, c) Solution A + B four volumes diluted with boiled distilled water. Each tectum was immersed 6 hr in 100 ml distilled water containing 0.5 g LiOH and 15 g KNO₃, washed 18 hr in 500 ml 0.2% acetic acid, dehydrated with ethanol, and embedded in low viscosity nitro cellulose. Sections were cut at 100 pm with a rotary microtome after clearing with cedarwood oil. Methods b) and c) have two advantages compared with method a), the original Golgi-Cox method. First, more cells are impregnated, especially in the layers extending 200-400 μm below the surface, and dendrites as well as unmyelinated axons are well impregnated. Second, myelin sheaths are impregnated and can be recognized by their peculiar chain-like appearance. The described Golgi-Cox modification offers an appropriate method to study the morphology of superficially located nervous tissue.     

Methyl Green-Pyronin for Staining Autoradiographs of Hydroxyethyl Methacrylate-Embedded Lymphoid Tissue

Cells in the spleen in DNA-synthesis were labelled with tritiated thymidine. Tissue was fixed for 12 hr in 10% neutral formalin, washed for 4 hr in tap water and dehydrated through 70% and absolute ethanol. The tissue blocks were infiltrated overnight with a mixture consisting of glycol methacrylate, 80 ml; polyethylene glycol 400, 12 ml; and benzoyl peroxide, 0.27 gm. Specimens were cast in BEEM capsules with the final embedding medium consisting of 42 parts of the infiltration medium and 1 part of an acceleration mixture. This mixture consisted of N,N-dimethylaniline, 1 part and polyethylene glycol 400, 15 parts. The blocks hardened in 30 min and were sectioned with an ultramicrotome fitted with glass knives. Sections were coated with Ilford K5 liquid emulsion and exposed for 2 wk. Methyl green-pyronin staining of autoradiographs was carried out at pH 4.1 in acetate buffer containing 0.5% methyl green (Allied Chemicals) and 0.2% pyronin GS (Chroma). Staining was for 30-60 min, after which sections were washed for 1 min in water, blotted, allowed to dry, and mounted in Canada balsm. The procedure resulted in good quality autoradiographs in which the degree of basophilia of labelled cells could be assessed.     

N-Butyl methacrylate and paraffin as an embedding medium for light microscopy.

A method of tissue embedding using n-butyl methacrylate and paraffin is described. Following alcohol dehydration and infiltration with the methacrylate monomer, tissues are embedded in gelatin capsules in a mixture consisting of 3.5 g of paraffin for each 10 ml of methacrylate. Benzoyl peroxide (0.2 g for each 10 ml of monomer) is added as the catalyst and the methacrylate polymerized in a 50 C oven for 18--24 h. Following polymerization the block is trimmed and embedded in paraffin to provide a firm support during sectioning. A water trough attached to the microtome knife is essential to facilitate the handling of sections and ribbons. For serial sections a mixture of equal weights of beeswax and paraffin is used to make the sections adhere to each other. Usual staining procedures can be used since the embedding medium is readily soluble in xylene.     

Counterstaining Golgi-Cox Impregnations with Luxol Fast Blue as a Myelin Stain

Immerse pieces of brain tissue 4 wk in solutions A and B, mixed just before use: A. K₂Cr₂O₇, 1 gm; HgCl₂, 1 gm; boiling distilled water, 85 ml. Boil A for 15 min, cool to 2 C and add: B. K₂CrO₄, 0.8 gm; Na₂WO₄, 0.5 gm; distilled water, 20 ml. Rinse in water and immerse 24 hr in LiOH, 0.5 gm; KNO₃, 15 gm; distilled water, 100 ml. Wash 24 hr in several changes of 0.2% acetic acid and then for 2 hr in tap water. Dehydrate and embed in celloidin. Process a 60 μ section through 70 and 95% ethanol, a 3:1 mixture of absolute ethanol and chloroform, and toluene. Immerse it for 5 min in a solution containing methyl benzoate, 25 ml; benzyl alcohol, 100 ml; chloroform, 75 ml. Orient the section on a chemically clean slide and let air-dry 5-10 min. Process through toluene, 3:1 ethanol-chloroform and 95% ethanol. Place the section for 5-60 min at 60 C in a solution made up of: Luxol fast blue G (Matheson, Coleman and Bell), 1 gm; 95% ethanol, 1000 ml; 10% acetic acid, 5 ml. Hydrate to water and immerse in 0.05% Li₂CO₃ for 3-4 min. Differentiate in 70% ethanol and place in water. Immerse for 5-15 min in a mixture of two solutions: A. cresylechtviolet (Otto C. Watzka, Montreal), 2 gm; 1 M acetic acid, 185 ml; B. 1 M sodium acetate, 15 ml; distilled water, 400 ml; absolute ethanol, 200 ml. Dehydrate to 3:1 ethanol-chloroform. Clear in toluene and apply a coverslip. The technique produces fast Golgi-Cox impregnated neurons against a background of counterstained myelinated fibers. Patterns of the myelinated fibers can be used to localize impregnated neurons.     

N-butyl Methacrylate and Paraffin as an Embedding Medium for Light Microscopy

A method of tissue embedding using n-butyl methacrylate and paraffin is described. Following alcohol dehydration and infiltration with the methacrylate monomer, tissues are embedded in gelatin capsules in a mixture consisting of 3.5 g of paraffin for each 10 ml of methacrylate. Benzoyl peroxide (0.2 g for each 10 ml of monomer) is added as the catalyst and the methacrylate polymerized in a 50 C oven for 18-24 h. Following polymerization the block is trimmed and embedded in paraffin to provide a firm support during sectioning. A water trough attached to the microtome knife is essential to facilitate the handling of sections and ribbons. For serial sections a mixture of equal weights of beeswax and paraffin is used to make the sections adhere to each other. Usual staining procedures can be used since the embedding medium is readily soluble in xylene.     

Staining and Mounting Helminths

The following procedure is recommended: Fix ces-todes and trematodes (while held flat between glass slides) 0.5-2.0 hr. in the following mixture: formalin, 15; acetic acid (gl.), 5; glycerol, 10; 95% ethyl alcohol, 24; distilled H₂O, 46; all proportions by volume. After freeing them from the slides, wash thoroughly in running water and stain immediately thereafter. Stock staining solution: ferric ammonium alum (violet cryst.), 2 g.; distilled H₂O (cold) 100 ml.; after solution, add 2 ml. concentrated H₂SO₄, bring to a boil; add 1 g. coelestin blue B (Nat. Aniline), boil 3-5 min.; cool and add 10 ml. absolute methyl alcohol and 10 ml. glycerol. Dilute 1 vol. with 3 vol. distilled H20 for use. Stain 5-30 min., depending on size of specimens. Wash with 2 changes 0.5 hr. each of distilled H₂O, then 50% isopropyl alcohol 12-16 hr., 50% isopropyl alcohol 2 hr., followed by graded isopropyl alcohol for dehydration. Ether: ethyl alcohol (equal parts), 1 hr., is followed by embedding in celloidin in a sheet just thick enough to cover the specimens. Trim embedded specimens and dehydrate with isopropyl alcohol, 80%, 90% and absolute. Clear in beechwood creosote. Mount in balsam with cover glasses that overlap the edges of the celloidin 1-2 mm. While drying at 37°C, refill edges of mount with fresh balsam as needed. When dry, remove excess balsam and ring the edges with ordinary gloss enamel paint.     

A method for staining pollen tubes in pistil

A quadruple staining procedure has been developed for staining pollen tubes in pistil. The staining mixture is made by adding the following in the order given: lactic acid, 80 ml; 1% aqueous malachite green, 4 ml; 1% aqueous acid fuchsin, 6 ml; 1% aqueous aniline blue, 4 ml; 1% orange G in 50% alcohol, 2 ml; and chloral hydrate, 5 g. Pistils are fixed for 6 hr in modified Carnoy's fluid (absolute alcohol:chloroform:glacial acetic acid 6:4:1), hydrated in descending alcohols, transferred to stain and held there for 24 hr at 45 +/- 2 C. They were then transferred to a clearing and softening fluid containing 78 ml lactic acid, 10 g phenol, 10 g chloral hydrate and 2 ml 1% orange G. The pistils were held there for 24 hr at 45 +/- 2 C, hydrolyzed in the clearing and softening fluid at 58 +/- 1 C for 30 min, then stored in lactic acid for later use or immediately mounted in a drop of medium containing equal parts of lactic acid and glycerol for examination. Pollen tubes are stained dark blue to bluish red and stylar tissue light green to light greenish blue. This stain permits pollen tubes to be traced even up to their entry into the micropyle.     

Leukocyte Culture and Chromosome Preparations from Adult Dog Blood

Plasma is obtained from dog blood after 3 hr settling in a syringe. Portions of the plasma (0.5-1.0 ml) are added to 4 ml of a medium consisting of 17 parts of BME Spinner, 3 parts of calf serum, 0.5 parts of glutamine, 0.5 parts of penicillin-streptomycin, and 0.1-1.0 parts of Scarlet Runner bean phytohemagglutinin. Colchicine, 0.1 ml of 10:1 stock solution, is added after 72 hr and incubation continued for 2 hr, then centrifuged 5 min at 700 rev/min. The supernatant is discarded, 3 ml of distilled water added, and the cell suspension centrifuged again. The supernatant is discarded and the fixative, consisting of 45% glacial acetic acid allowed to act for 0.5 hr. Acetic-orcein stains of smears were very satisfactory.     

Binding of C3 and C3dg to the CR2 complement receptor induces growth of an Epstein-Barr virus-positive human B cell line

The effect of ligand interactions with the C3d/C3dg complement receptor (CR2) on proliferation of human B lymphoblastoid cells was investigated by using cell cultures performed at low density (1 to 1.5 x 10(3) cells/ml) in a serum-free defined medium to which only transferrin had been added. This medium does not allow proliferation of Raji cells which die within 48 hr with formation of polykaryons. Addition of purified human C3 to the cultures resulted in a dose-dependent proliferation of the cells. A steady growth of Raji cells with a doubling time of 36 hr was observed in cultures containing 10 micrograms/ml of C3. A growth rate similar to that observed in the presence of native C3 was found in the presence of equimolar concentrations of purified C3dg but not of C3c. F(ab')2 anti-C3d but not F(ab')2 anti-C3c antibodies inhibited the mitogenic effect of C3. Preincubation of Raji cells with monoclonal antibody OKB7 which directly inhibits the binding of C3dg to CR2, totally suppressed C3-induced growth of the cells. C3 did not enhance growth of the T lymphoma-derived cell line JM and monocytic cell line U937 which do not express CR2. These results provide direct evidence that the interaction between CR2 and C3 fragments stimulates proliferation of human cells of the B lineage. Because CR2 also acts as a receptor for Epstein-Barr virus on B cells, our results may pertain to the B cell mitogenic properties of the virus.     

Effect of 2-hydroxyethyl methacrylate on Streptococcus spp. biofilms

Aims: The effect of different concentrations of 2‐hydroxyethyl methacrylate (HEMA) was evaluated on biofilm formation and preformed biofilm of Streptococcus mitis, Streptococcus mutans and Streptococcus oralis, alone or combined to each other. Methods and Results: Twofold serial dilution of HEMA ranged from 12 to 0·75 mmol l^?1 was added to Streptococcal broth cultures and mature biofilms in 96‐well‐microtitre plates to evaluate bacterial biomass and cell viability. HEMA affected the Streptococcal population in a strain‐specific way producing few significant effects. A reduction on biofilm formation and a detachment of preformed biofilm was recorded in Strep. mitis ATCC 6249, whereas in mixed cultures, the monomer expressed a general aggregative effect on mature biofilms. A reduction in cell viability was also recorded in an HEMA‐concentration‐dependent way in each experimental condition studied. Conclusions: These results suggest that the HEMA prevalent effects are both the reduction of bacterial adhesion to a polystyrene surface and the increase in dead cells also characterized by an aggregative status. Significance and Impact of the Study: Understanding the potential effect of HEMA, released from resin‐based materials, on oral bacteria may furnish information for surveillance of the risk reduction in secondary caries via hindering biofilm generation.     

A Tungstate Modification of the Golgi-Cox Method

Pieces of fresh nervous tissue 4-5 mm thick are put into the following solution: HgCl₂, 1 gm; K₂Cr₂O₇, 1 gm; K₂CrO₄, 0.8 gm; K₂WO₄ (or Na₂WO₄), 0.5 gm; distilled water 100 ml. They are kept undisturbed in the dark at room temperature for 20-30 days, then transferred to the following alkaline solution: LiOH (or NaOH), 1 gm; KNO₃, 15 gm; distilled water, 100 ml. After 12-24 hr in this solution they are washed for 12-24 hr in several changes of distilled water. (If sodium hydroxide was used, 0.5 ml of acetic acid should be added per 100 ml of wash water.) Embedding in celloidin follows dehydration. Sections are dehydrated in 3 parts of absolute alcohol and 1 part of chloroform, cleared in iodobenzene and mounted with a cover slip using a mounting medium with a refractive index around 1.61. The use of tungstate improves the general results and allows especially successful impregnations in very young animals, when the usual technic fails.     

A Single solution iron-hematoxylin Stain for Intestinal Protozoa

A single solution iron-hematoxylin stain is described for staining fecal smears rapidly and simply. The stain is prepared from the following solutions: Solution A: 1% hematoxylin in 95% alcohol, prepared by diluting a stock solution of 10% hematoxylin in 95% alcohol. Solution B: Ferric ammonium sulfate (violet crystals), 4.0 g.; glacial acetic acid, 1.0 ml.; concentrated sulfuric acid (sp. gr. 1.8),0.12 ml.; distilled water, 100 ml. Mix equal parts of Solution A and Solution B; allow to stand overnight, filter and use. For maximum length of staining life, store in full, air-tight bottles. To stain fecal smears, fix in Schaudinn's, pass through iodine alcohol to 50% alcohol, stain for three minutes, wash in running tap water 5 to 15 minutes, dehydrate and mount.     

A Single solution iron-hematoxylin Stain for Intestinal Protozoa

A single solution iron-hematoxylin stain is described for staining fecal smears rapidly and simply. The stain is prepared from the following solutions: Solution A: 1% hematoxylin in 95% alcohol, prepared by diluting a stock solution of 10% hematoxylin in 95% alcohol. Solution B: Ferric ammonium sulfate (violet crystals), 4.0 g.; glacial acetic acid, 1.0 ml.; concentrated sulfuric acid (sp. gr. 1.8),0.12 ml.; distilled water, 100 ml. Mix equal parts of Solution A and Solution B; allow to stand overnight, filter and use. For maximum length of staining life, store in full, air-tight bottles. To stain fecal smears, fix in Schaudinn's, pass through iodine alcohol to 50% alcohol, stain for three minutes, wash in running tap water 5 to 15 minutes, dehydrate and mount.     

Improved developmental competence of cloned porcine embryos with different energy supplements and chemical activation

The present study investigated the effect of lactate/pyruvate supplement in culture medium and of chemical activation after electric stimulus on in vitro development of porcine somatic cell nuclear transfer (SCNT) embryos. In vitro matured gilt oocytes were enucleated, reconstructed with fetal fibroblasts, and simultaneously fused/activated using a single pulse of 2.0 kV/cm for 30 microsec. In Experiment 1, reconstructed embryos were cultured in North Carolina State University (NCSU)-23 medium supplemented with either 5.5 mM glucose (Group A) or lactate (5.0 mM)/pyruvate (0.5 mM) (Group B). Compared to Group A, cleavage rate (64% vs. 47%) was higher and more blastocysts developed in Group B (17% vs. 6% at Day 6, 21% vs.11% at Day 7). Experiment 2, embryos reconstructed by electric stimulus (2.0 kV/cm for 30 microsec) were subjected to three activation protocols: (1) no chemical activation (Group C), (2) 7.5 microg/ml cytochalasin B treatment at 2 hr after electric stimulus (Group D), and (3) 5 microg/ml 6-dimethylaminopurine (Group E) treatment at 2 hr after electric stimulus. The reconstructed embryos were cultured for 7 days in NCSU-23 medium supplemented with lactate (5.0 mM)/pyruvate (0.5 mM). The rates of blastocyst formation on Day 6 and Day 7 in Group C (17 and 20%, respectively) or Group D (15, 20%, respectively) were higher than in Group E (9 and 12%, respectively). The percentage of two pseudo-pronucleus (PPN) formations in Group D (88%) was significantly higher than in Group C (71%) and Group E (72%). Mean cell numbers of blastocysts in Group D (63.4 +/- 15.8) were higher than in Group C (43.9 +/- 16.5) and Group E (32.9 +/- 17.9), due to increased trophectoderm (TE) cell numbers. Our results indicate that supplementing NCSU-23 medium with lactate/pyruvate and exposure of cytochalasin B after electrical stimulus can improve in vitro developmental competence of porcine SCNT embryos.     

Effects of prostaglandin E2 and F2 alpha on cytoplasmic pH in a clonal osteoblast-like cell line, MOB 3-4.

Prostaglandin E2 (PGE2, 5 ng/ml to 5 micrograms/ml) induced a dose-dependent increase in cAMP accumulation, inositol phosphates (IPs) accumulation, and cytoplasmic free Ca2+ ([Ca2+]i) in a clonal osteoblast-like cell line, MOB 3-4. In contrast, prostaglandin F2 alpha (PGF2 alpha, 5 ng/ml to 5 micrograms/ml) stimulated increases in IPs accumulation and [Ca2+]i without stimulating an increase in cAMP accumulation. Both PGE2 (greater than 0.5 micrograms/ml) and PGF2 alpha (greater than or equal to 5 micrograms/ml) increased cytoplasmic pH (pHi) from approximately 7.15 to 7.35 in BCECF-loaded cells. A tumor promotor, phorbol 12-myristate 13-acetate (PMA, 0.1-100 nM) also increased pHi without effect on phosphoinositide hydrolysis. Both PGE2-(5 micrograms/ml) and PMA- (100 nM) induced cytoplasmic alkalinization was inhibited by removal of extracellular Na+, or by pretreatment of the cells with amiloride (0.5 mM), an inhibitor of Na+/H+ exchange, or H-7 (100 microM), a nonspecific inhibitor of protein kinase C. Thus, MOB 3-4 cells appeared to possess PGE2 receptors and PGF2 alpha receptors: the former are coupled to adenylate cyclase and phospholipase C, and the latter are predominantly coupled to phospholipase C. Also the cells appeared to possess an amiloride-sensitive Na+/H+ exchange activity, which increases pHi in response to PGE2 and PGF2 alpha, as well as to PMA. Long-term (48 hr) exposure of the cells to PGE2 at a high concentration (5 micrograms/ml), but not to PGF2 alpha and PMA, decreased DNA synthesis in the serum-deficient medium. Thus, cytoplasmic alkalinization appeared insufficient for cell replication. At least in MOB 3-4 cells, the inhibitory effect of PGE2 on DNA synthesis may be due to the cAMP messenger system.     

Cell-culturing characteristics of newly developed PHEMA microcarriers: their use with BHK21 cells.

Baby hamster kidney (BHK) fibroblasts, as model cells, have been proliferated on acrylic based microcarriers. Microcarriers were prepared by a novel suspension polymerization of acrylic monomers. Hydroxyethyl methacrylate was the basic monomer. Ethylene glycol dimethacrylate was used as the cross-linker. A hydrophobic comonomer, namely, methyl methacrylate, was included in order to adjust the hydrophilicity of the resultant matrix. An acrylic comonomer with positively charged tertiary amine groups, i.e., dimethylaminoethyl methacrylate, was also added in order to optimize the surface charge of the carriers. The adhesion, spreading, and growth characteristics of BHK cells on these novel beads were studied either in stationary or in submerged culture conditions. The results demonstrate that the cell attachment and growth can be controlled by changing the degree of charge and the hydrophilicity of the poly(hydroxyethyl methacrylate) matrix.