Bio-functionalized thermoresponsive interfaces facilitating cell adhesion and proliferation

Bio-functionalized thermoresponsive culture interfaces co-immobilized with cell adhesive peptide, RGDS, and cell growth factor, insulin (INS), are investigated to promote initial cell adhesion and cell growth for further cell sheet engineering applications. These bio-functionalized interfaces were prepared by electron beam-induced copolymerization of N-isopropylacrylamide (IPAAm) with its carboxyl-derivatized analog, 2-carboxyisopropylacrylamide (CIPAAm), and grafting onto tissue culture polystyrene dishes, followed by immobilization of RGDS and/or INS to CIPAAm carboxyls. Adhesion and proliferation of bovine carotid artery endothelial cells (ECs) were examined on the RGDS-INS co-immobilized thermoresponsive interfaces. Immobilized RGDS facilitated initial EC adhesion on the surfaces and INS modification was demonstrated to induce EC proliferation, respectively. More pronounced EC growth was indicated by co-immobilization of appropriate amount of RGDS and INS. This may be due to synergistic effect of direct co-stimulation of adhered ECs by surface-immobilized RGDS and INS molecules. ECs grown on the RGDS-INS co-immobilized thermoresponsive interfaces can also be recovered spontaneously as viable tissue monolayers by solely reducing culture temperature. RGDS-INS co-immobilized thermoresponsive interfaces strongly supported initial EC adhesion and growth than unmodified thermoresponsive surfaces even under serum-free culture. Addition of soluble growth factors to serum-free culture medium effectively induced EC proliferation to confluency. Co-immobilization of cell adhesion peptides and growth factors on thermoresponsive surfaces should be effective for rapid preparation of intact cell sheets and their utilization to regenerative medicine.     

Biomodification of non-woven polyester fabrics by insulin and RGD for use in serum-free cultivation of tissue cells

In this study, the development of a novel cell support material was purposed as due to the serum-free cultivation of tissue cells. This material was prepared by immobilizing RGD (Arg-Gly-Asp) sequence of cell-adhesion factor, fibronectin, and cell-growth factor, insulin, to the three-dimensional non-woven polyester fabric (briefly NWPF) discs that have been used successfully in our previous cell culture studies. At first these matrices were partially hydrolyzed and then the carboxyl groups were coupled with RGD or insulin in the presence of water-soluble carbodiimide. The effectiveness of immobilization process was checked with SEM, ATR-FTIR spectroscopy and swelling studies. The maximum amount of immobilized insulin was 6.96 micorgcm(-2) and it was obtained at 200 micorgml(-1) initial insulin concentration for 60 min immobilization period. The cell culture studies which were carried out with human skin fibroblasts (HS An1) showed that, percentage of adhesion on RGD modified NWPF discs is higher than that of other surfaces. i.e., unmodified discs, polystyrene Petri dishes and insulin-immobilized discs, in serum-free culture. According to the results of growth studies, highest cell yield was obtained in the case of insulin-modified discs.     

Decrease in culture temperature releases monolayer endothelial cell sheets together with deposited fibronectin matrix from temperature-responsive culture surfaces.

Bovine aortic endothelial cells were cultured on surfaces grafted with a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), in the presence of serum. Cells adhered, spread, proliferated, and reached confluency as observed on ungrafted tissue culture polystyrene dishes. A decrease in culture temperature released cells only from the grafted surfaces without enzymatic or ethylenediaminetetraacetic acid treatment. Upon lowering temperature, the culture surfaces changed from hydrophobic to hydrophilic owing to the hydration of grafted PIPAAm and thus weakened the cell attachment to the dishes. Released cells maintained cell-cell junctions composing monolayer cell sheets. Immunoblotting and immunofluorescence microscopy revealed that fibronectin (FN) was deposited and accumulated on the grafted surfaces during the culture. Furthermore, the deposited FN matrix adhering to cell sheets was also recovered from temperature-responsive surfaces by low-temperature treatment, while trypsin treatment destroyed the matrix. The recovery of FN by low-temperature treatment was as high as by physical scraping with a rubber blade. Temperature-responsive surfaces can provide a novel method to use cultured confluent cell sheets for tissue engineering, and also to elucidate structure and function of deposited extracellular matrix during cell culture.     

Immobilization of cell-adhesive peptides to temperature-responsive surfaces facilitates both serum-free cell adhesion and noninvasive cell harvest

We have developed temperature-responsive cell culture surfaces to harvest intact cell sheets for tissue-engineering applications. Both cost and safety issues (e.g., prions, bovine spongiform encephalopathy) are compelling reasons to avoid use of animal-derived materials, including serum, in such culture. In the present study, synthetic cell-adhesive peptides are immobilized onto temperature-responsive polymer-grafted surfaces, and cell adhesion and detachment under serum-free conditions were examined. The temperature-responsive polymer poly(N-isopropylacrylamide) (PI-PAAm) was functionalized by copolymerization with a reactive comonomer having both a carboxyl group and an isopropylacrylamide group. These copolymers were covalently grafted onto tissue culture-grade polystyrene dishes. Synthetic cell-adhesive peptides were then immobilized onto these surfaces via carboxyl groups. Bovine aortic endothelial cells both adhered and spread on these surfaces even under serum-free conditions at 37 degrees C, similar to those in 10% serum-supplemented culture. Spread cells promptly detached from the surfaces on lowering culture temperatures below the lower critical solution temperature of the polymer, 32 degrees C. These surfaces would be useful for serumfree culture for tissue-engineering applications.     

Two-dimensional cell sheet manipulation of heterotypically co-cultured lung cells utilizing temperature-responsive culture dishes results in long-term maintenance of differentiated epithelial cell functions.

Here we report two-dimensional cell sheet manipulation (2D CSM) of heterotypically co-cultured lung cell sheets and the maintenance of differentiated phenotypes of lung epithelial cells over prolonged periods of up to 70 days. This was facilitated by poly(N-isopropylacrylamide) (PIPAAm)-grafted tissue culture dishes. PIPAAm-grafted dishes are responsive to temperature changes and offer a unique surface on which cells adhere and multiply like on ordinary tissue culture dishes under the permissive temperature of 37 degrees C, but on lowering of temperature resulting in changes in hydration of the polymer the cells spontaneously detach from the surface without use of enzymes like trypsin which is the common procedure. It has been well documented that type II pneumocytes of the lung lose many of their special features rapidly in culture. The culture system detailed here comprises random co-culture of epithelial and mesenchymal cells of lung. The heterotypic cell culture system promotes cell-cell interactions maintaining a harmonized physiology. When this heterotypic monolayer on PIPAAm-grafted dishes was subjected to lower temperature of 20 degrees C and 2D CSM we were able to transfer the monolayer as a single contiguous sheet with cell-cell connections intact to other surfaces. This non-invasive transfer of cell sheet resulted in shrinkage of the monolayer, enabling the type II cells to regain their cuboidal morphology and specialized characters like Maclura pomifera lectin binding and surfactant protein A (SP-A) expression. The active dome formation also observed subsequent to transfer reaffirms the uniqueness of the culture conditions and 2D CSM in future for developing tissue like architecture in vitro.     

Structural characterization of bioengineered human corneal endothelial cell sheets fabricated on temperature-responsive culture dishes

For the purpose of corneal regenerative medicine, we fabricated human corneal endothelial cell sheets on temperature-responsive dishes, which could be non-invasively harvested as intact, transplantable sheets by simply reducing the culture temperature. Cells demonstrated hexagonal cell shape with numerous microvilli and cilia, and also exhibited abundant cytoplasmic organelles similar to these cells in vivo. Immunofluorescence for type IV collagen and fibronectin revealed that abundant extracellular matrix (ECM) was deposited on the basal surface throughout culture, and the deposited ECM was harvested along with the cell sheets by reducing culture temperature to 20 degrees C. Faint ECM remnants were observed on the dish surfaces after cell sheet detachment. Immunofluorescence for ZO-1 showed that tight junctions were established between cells, and immunoblotting indicated that intact ZO-1 was maintained during cell sheet harvest, while conventional proteolytic cell harvest methods resulted in the degradation of ZO-1. These results suggest that these transplantable corneal endothelial cell sheets can be applied to treat patients with damaged corneas.     

Transplantable urothelial cell sheets harvested noninvasively from temperature-responsive culture surfaces by reducing temperature

Augmentation cystoplasty using gastrointestinal flaps may induce severe complications such as lithiasis, urinary tract infection, and electrolyte imbalance. The use of viable, contiguous urothelial cell sheets cultured in vitro should enable us to avoid these complications. Transplantable urothelial cell sheets were obtained by utilizing a temperature-responsive cell culture method, and then examined by immunostaining and electron microscopy. Canine urothelium was produced on the surfaces of temperature-responsive culture dishes covalently bonded with the thermally sensitive polymer, poly(N-isopropylacrylamide). Stratified urothelial cell sheets were cultured and then harvested intact without enzymatic treatment from these dishes by reducing the temperature. Histological structure and cell-to-cell junctions were compared between these urothelial cell sheets and those harvested with dispase. All urothelial cell sheets were harvested from the bonded surfaces by reducing the culture temperature without the need for dispase. Electron microscopy revealed well-developed microridge, microvilli, and cell junction complexes. Conversely, these same cell features were destroyed by dispase treatment. Immunoblotting revealed that dispase fragmented occludin, whereas it remained unchanged in the intact urothelial cell sheets. Novel urothelial cell sheets obtained by culture on temperature-responsive culture surfaces were successfully harvested much less destructively than with dispase. This technology should prove useful in urinary tract tissue engineering in the near future.     

Temperature-responsive culture dishes allow nonenzymatic harvest of differentiated Madin-Darby canine kidney (MDCK) cell sheets

We have developed a temperature-responsive culture dish grafted with a poly(N-isopropylacrylamide) (PIPAAm). Various types of cells adhere, spread, and proliferate on the grafted dishes in the presence of serum at 37 degrees C. By reducing only temperature, these cells can be harvested noninvasively from the dishes according to rapid hydration of the grafted polymer. Because the harvest does not need enzymatic digestion, differentiated cell phenotypes are retained. In the present study, a renal epithelial cell line, Madin-Darby canine kidney (MDCK) cell, was cultured on the dishes, and cell behavior was examined. MDCK cells showed differentiated phenotypes such as dome formation during long-term culture, similar to on ungrafted dishes. After 1-week culture at 37 degrees C, trypsin digestion disrupted cell-cell junctions but failed to liberate cells from both ungrafted and grafted dishes. However, short-term incubation at 20 degrees C released confluent MDCK cells as a single contiguous cell sheet only from the polymer-grafted dishes because of selective disruption of the cell-surface binding. Immunocytochemistry with anti-beta-catenin antibody revealed that functional cell-cell junctions were organized even in the recovered cell sheets. Intriguingly, incubation time at 20 degrees C required for cell sheet detachment gradually shortened during long-term culture before reducing temperature. The acceleration of cell detachment was correlated to the decrease of a single cell area by means of cell contractile force. These findings suggest that cell sheet detachment from PIPAAm-grafted dishes should be accomplished by both PIPAAm hydration and cellular metabolic activity such as cell contraction.     

Temperature-Responsive surface for novel co-culture systems of hepatocytes with endothelial cells: 2-D patterned and double layered co-cultures

We have developed two novel cell co-culture system, without any on cell type combination limitation, utilizing a polymer surface which is temperature-sensitive with respect to its cell adhesion characteristics. One system involves a patterned co-culture of primary hepatocytes with endothelial cells utilizing patterned masked of the electron-beam cured, temperature-responsive polymer, poly (N-isopropylacrylamide) (PIPAAm) by masked electron beam irradiation. Hepatocytes were cultured to confluency at 37 degrees C on these surfaces. When the culture temperature was reduced below 32 degrees C, cells detached from the PIPAAm-grafted areas without any need for trypsin. Endothelial cells were then seeded onto the same surfaces at 37 degrees C. These subsequently seeded endothelial cells adhered only to the now-exposed PIPAAm-grafted domains and could be co-cultured with the hepatocytes initially seeded at 37 degrees C in well-ordered patterns. The other system involves a double layered co-culture obtained by overlaying endothelial cell sheets of the designed shape onto hepatocyte monolayers. The endothelial cells adhered and proliferated on the PIPAAm-grafted surface, as on polystyrene tissue culture dishes at 37 degrees C. By reducing the temperature, confluent monolayers of cells detached from the PIPAAm surfaces without trypsin. Because the recovered cells maintained intact cell-cell junctions together with deposited extracellular matrix, the harvested endothelial cell sheets, with designed shapes, were transferable and readily adhered to hepatocyte monolayers. Stable double layered cell sheets could be co-cultivated. These two co-culture methods enabled long-term co-culture of primary hepatocytes with endothelial cells. Hepatocytes so co-cultured with endothelial cells maintained their differentiated functions, such as albumin synthesis for unexpectedly long periods. These novel two co-culture systems offer promising techniques for basic biologic researches upon intercellular communications, and for the clinical applications of tissue engineered constructs.     

Characterization of human esophageal carcinoma cell line established on confluent monolayer, and advantage of confluent monolayer surface structure for attachment and growth

Biopsy tissue from a 72-year-old Japanese woman with metastasis to the cervical lymph nodes of recurrent esophageal carcinoma was adapted for culturing on the surface of lethally X-irradiated confluent monolayers of Balb/c 3T3 cells, and a continuous-growing tumor cell line was developed. These cells were found to contain keratinous material by immunohistochemical staining. We confirmed that these cells were of human origin by performing chromosome analysis. Thus, this line, designated as KSE-2 line, was considered to have originated from metastatic squamous-cell carcinoma of the human esophagus. When the attachment and growth of KSE-2 was examined on various culture surfaces, the confluent monolayers of Balb/c 3T3 cells were found to be more suitable than other substrata, such as plastic and collagen-coated dishes. The fixation of confluent monolayers of 3T3 cells with 3% glutaraldehyde had almost no affect on the colony-forming efficiency of the KSE-2 cells. In addition, the period of lag phase after inoculation of KSE-2 cells on fixed as well as nonfixed monolayers was shorter than that on plastic dishes. The conditioned medium from lethally X-irradiated 3T3 cells did not enhance the attachment and proliferation of KSE-2 cells on both nonfixed and fixed confluent monolayers. From these results, the surface structure itself of the 3T3 confluent monolayer was considered to play an important role in the favorable attachment and proliferation of KSE-2 cells.     

Culture and recovery of macrophages and cell lines from tissue culture-treated and -untreated plastic dishes

Macrophages can be separated from other cell types by their ability to readily attach and spread on glass or on plastic surfaces which are treated for optimal growth of cultured cells (tissue culture-treated plastic). To detach macrophages from these surfaces, techniques must be used which require prior preparation of special flasks or vessels, utilize expensive equipment, are time-consuming and almost uniformly require that the macrophages be exposed to various chemicals. We now report that macrophages can be enriched and recovered efficiently after attachment to disposable polystyrene bacteriologic petri dishes simply by gentle scraping with a rubber policeman. In this paper we compare this method to others currently in use in which resident peritoneal cells, peritoneal exudate cells or cells from bone marrow-derived cultures are detached from treated dishes using cold shock, chelating agents and lidocaine. In all studies, advantages were noted when cells were incubated in untreated dishes and detached by gentle scraping. In addition, untreated dishes supported the growth of adherent cell lines IC-21 and L929B and yielded large numbers of cells, with high viability, which were easily harvested.     

Two-dimensional manipulation of cardiac myocyte sheets utilizing temperature-responsive culture dishes augments the pulsatile amplitude

Although cardiac myocytes adherent to tissue culture polystyrene (TCPS) dishes retain the spontaneous beating, the pulsatile amplitude is highly limited compared to that in vivo. One of the main reasons for the limited pulsation may be the interface between the cells and the TCPS surfaces. Release of these cells from rigid TCPS surfaces may augment their pulsatile amplitude. With this perspective, we have developed a novel cell manipulation technique to detach cultured cardiac myocytes from rigid surfaces and to rescue higher pulsatile amplitude of the cells using temperature-responsive culture dishes and discuss the possibility of improving this heart tissue model. Primary cardiac myocytes were cultured on the slightly hydrophobic dish surfaces grafted with a temperature-responsive polymer, poly(N-isopropylacrylamide). Cells adhered and proliferated, forming confluent cardiac myocyte sheets in a fashion similar to those on ungrafted TCPS dishes. Decrease in culture temperature resulted in surface change of the polymer from slight hydrophobic to highly hydrophilic due to extensive hydration of the grafted polymer on the dishes. This results in release of cardiac myocyte sheets from the dishes without enzymatic or EDTA treatment. When no support was used, the detached cardiac myocyte sheets shrank to one-tenth size, which ceased their pulsation. When chitin membranes were used to support the confluent sheets to prevent cell shrinkage, the detached cell sheets could be transferred and readily adhered onto another virgin TCPS dishes. These transferred cell sheets preserved the similar cell morphology and pulsation to those before the detachment. When polyethylene meshes were used to support cell sheet transfer, detached cardiac myocyte sheets partially attached to the mesh threads. Then, the constructs were inverted and placed in another culture dish to prevent direct association to dish surfaces. Moreover, the cardiac myocyte sheets were reorganized to heart tissue-like structures by the unisotropic contraction orientated by the mesh threads, and the pulsatile amplitude increased more than 10 times higher. This technique would bring about new insight in tissue engineering as well as cultured heart model.     

Cell growth on immobilized cell-growth factor. II. Adhesion and growth of fibroblast cells on poly(methyl methacrylate) membrane immobilized with proteins of various kinds.

The surface of poly(methyl methacrylate) membrane was partially hydrolysed and the carboxyl groups produced were coupled with various protein molecules with water-soluble carbodiimide. The immobilized proteins were a cell-growth factor insulin, cell adhesion factors fibrinogen and fibronectin, and serum proteins albumin and gamma-globulin. The insulin-immobilized poly(methyl methacrylate) membrane strongly accelerated the growth and slightly accelerated the adhesion of fibroblast cells. The immobilized fibronectin and fibrinogen enhanced the cell adhesion, and the former also accelerated the cell growth. The immobilized albumin and gamma-globulin influenced the adhesion and growth of cells very little. It was found that various proteins specifically influence the adhesion and growth of cells in an immobilized state.     

Cell growth on immobilized cell growth factor. I. Acceleration of the growth of fibroblast cells on insulin-immobilized polymer matrix in culture medium without serum.

Culture of fibroblast cells in serum-free medium in the presence of insulin-immobilized films of poly(ethylene terephthalate) or poly(methyl methacrylate) accelerated the cell growth to several times that in the presence of free insulin molecules. The immobilization of insulin was useful also for elucidation of the mechanism of signal transmission through receptor molecules in the cell membrane. Free insulin molecules are known to be bound by receptors and the complex formed internalized and degraded in the cytoplasm. However, it is not clear what process is indispensable for transmission of the biological signals to the nucleus of the cell. The present investigation revealed that cell growth was enhanced by the addition of immobilized insulins which were not internalized. It appears that insulin-receptor binding is enough for transmission of the cell growth signal to the cell nucleus. The enhanced cell growth by the immobilized insulins compared with free insulin molecules is ascribable to suppression of the down-regulation which is a consequence of intracellular decomposition of the insulin-receptor complex.     

Expression of mucin synthesis and secretion in human tracheobronchial epithelial cells grown in culture

The effects of culture conditions on growth and differentiation of human tracheobronchial epithelial (HTBE) cells have been defined. Epithelial cells were dissociated from tissues by protease treatment and were plated on tissue culture dishes in F12 medium supplemented with insulin, transferrin, epidermal growth factor, hydrocortisone, cholera toxin, bovine hypothalamus extract, and retinol. HTBE cells did not express any mucociliary function (ciliogenesis or mucin secretion) on tissue culture plastic, but they could be passaged 3 to 5 times with a total of 10 to 25 population doublings. Cells from early passages re-express both these functions when transplanted to tracheal grafts. When tissue culture plates were coated with collagen film or collagen gel substrata, cell attachment and proliferation were stimulated. However, the expression of mucous cell function in culture occurred only when cells were plated on collagen gel substrata and vitamin A (retinol) was present in the medium. Mucous cell differentiation under optimal conditions was defined by ultrastructural studies, by immunologic studies with mucin-specific monoclonal antibodies, and by carbohydrate and amino acid compositional analyses of mucin-like glycoproteins purified from culture medium. These results demonstrate for the first time that HTBE cells can express mucin synthesis and secretion under appropriate culture conditions.     

Patterned biofunctional designs of thermoresponsive surfaces for spatiotemporally controlled cell adhesion, growth, and thermally induced detachment

In the present study, we report advanced patterned biofunctionalization of thermoresponsive surfaces for achievement of spatiotemporally controlled cell adhesion, growth, and thermally induced detachment. These patterned biofunctional thermoresponsive surfaces were prepared using dual surface modification techniques: electron beam-induced surface patterning of carboxyl-functional thermoresponsive polymers with appropriate metal masks and following site-selective biofunctionalization with biomolecules, the cell adhesive peptide (RGDS) and/or the cell growth factor (insulin; INS). Patterned co-immobilization of RGDS-INS onto thermoresponsive surfaces dominated site-selective cell adhesion and growth along with patterned biofunctional domains in the serum-free culture. Spatiotemporal detachment of sparsely adherent and confluent cells from these patterned biofunctional thermoresponsive surfaces were both achieved only by reducing temperature. Furthermore, RGDS-INS-patterned thermoresponsive surfaces also successfully demonstrated the selective fabrication and recovery of either contiguous monolayer or mesh-like designed monolayer tissue constructs on the identical surfaces. Thus, patterned biofunctional designs would be utilized for the creation and harvest of biomimetic-designed vascular networks having sufficient biofunctional activities in facilitated cell sheet engineering and regenerative medicine.     

A self-contained microfluidic cell culture system

Conventional in vitro cell culture that utilizes culture dishes or microtiter plates is labor-intensive and time-consuming, and requires technical expertise and specific facilities to handle cell harvesting, media exchange and cell subculturing procedures. A microfluidic array platform with eight microsieves in each cell culture chamber is presented for continuous cell culture. With the help of the microsieves, uniform cell loading and distribution can be obtained. Within the arrays, cells grown to the point of confluency can be trypsinized and recovered from the device. Cells trapped in the microsieves after trypsinization function to seed the chambers for subsequent on-chip culturing, creating a sustainable platform for multiple cycles. The capability of the microfluidic array platform was demonstrated with a BALB/3T3 (murine embryonic fibroblast) cell line. The present microfluidic cell culture platform has potential to develop into a fully automated cell culture system integrated with temperature control, fluidic control, and micropumps, maximizing cell culture health with minimal intervention.     

Enhanced effect of sulfonylurea (SU) in copolymer comprising a sugar moiety and SU derivative as double ligands on insulin secretion from MIN6 cells

Copolymers composed of sulfonylurea (SU) as an antagonist of ATP-sensitive K+ channel and sugar moieties as double ligands were synthesized. Insulin secretion from MIN6 cells (insulinoma cell line) in contact with the copolymers was evaluated. MIN6 cells attached to the poly(N-p-vinylbenzyl-D-maltonamide-co-SU) [P(VMA-co-SU)]-coated dishes were in the more aggregated form as compared to other polymer-coated surfaces. By introducing SU into the sugar bearing homopolymer, an enhanced effect on insulin secretion from MIN6 cells was observed due to the specific interaction between SU ligands and SU receptors on the beta-cell membrane. P(VMA-co-SU) composed of SU and non-reducing glucose moieties demonstrated enhanced insulin secretion from MIN6 cells and faster proliferation of MIN6 cells as compared to poly(N-p-vinylbenzyl-D-lactonamide-co-SU) [P(VLA-co-SU)] probably owing to the glucose transporters presence on the MIN6 cell membrane. Insulin secretion from MIN6 cells pretreated with diazoxide as an agonist of ATP-sensitive K+ channel was suppressed.     

Immune response to immobilized sheep erythrocyte monolayer

Spleen cells from adult specific pathogen-free miniature swine were exposed to sheep red blood cell (SRBC)² monolayers to follow the course of the immune response to immobilized particulate antige. Spleen cells in medium were incubated for various time intervals on the monolayers, removed and cultured in new dishes without further exposure to SRBC antigen for 5.5 days. Spleen cells from unimmunized ‘normal’ animals responded maximally with 6-h exposure to a monolayer consisting of 75% SRBC and 25% autologous pig red blood cells. Spleen cells from SRBC primed animals also responded optimally to monolayers of 75% SRBC but required only s-h exposure before culturing. Only minimal numbers of SRBC were released from the monolayers, and carryover of antigen from the monolayer to the fresh tissue culture dishes was not a factor in promoting response. This method of in vitro immunization provides for pulse exposure to particulate antigen and facilitates precise evaluation of the role of antigen during the course of the immune response.     

Purification and quantification of T and B lymphocytes by an affinity method.

Affinity surfaces were produced by coupling human immunoglobulin (HGG) to the surface of tissue culture grade plastic-ware with a water-soluble carbodiimide followed by treatment with anti-HGG antisera. Surface immunoglobulin SIg) bearing human B lymphocytes attach to these surfaces when centrifuged on to them and unattached cells could be recovered by inverting the trays or dishes. Optimal cell attachment conditions could be rapidly evaluated by counting cells attached to representative areas of multi-well trays and percentage of SIg-bearing cells quantified. Evidence was obtained for cell attachment through Fc receptors as well as SIg using unrelated antigen--antibody-coated trays. This could be prevented by using the F (ab')2 fragments of the antisera. Under these conditions specific attachment through K and lambda light chains could be achieved with normal and chronic lymphocytic leukaemic lymphocytes. Using tissue culture plastic Petri dishes and relatively small quantities of antiserum, larger numbers of lymphocytes could be processed to produce T lymphocytes containing less than 1 per cent of contaminating SIg-positive cells.