The molecular weight and concentration of dextran sulfate affect cell growth and antibody production in CHO cell cultures

To investigate the effect of dextran sulfate (DS), a widely used anti‐aggregation agent, on cell growth and monoclonal antibody (mAb) production including the quality attributes, DS with the three different MWs (4,000 Da, 15,000 Da, and 40,000 Da) at various concentrations (up to 1 g/L) was added to suspension cultures of two different recombinant CHO (rCHO) cell lines producing mAb, SM‐0.025 and CS13‐1.00. For both cell lines, the addition of DS, regardless of the MW and concentration of DS used, improved cell growth and viability in the decline phase of growth. However, it increased mAb production only in the CS13‐1.00 cells. Among the three different MWs, 40,000 Da DS was most effective in attenuating cell aggregation during the cultures of CS13‐1.00 cells, and showed the highest maximum mAb concentration. For SM‐0.025 cells, it significantly decreased specific mAb productivity, particularly at a high concentration of DS. Overall, DS addition did not negatively affect the quality attributes of mAbs (aggregation, charge variation, and glycosylation), though its efficacy on mAb quality depended on the MW and concentration of DS and cell lines. For both cell lines, the addition of DS did not affect N‐glycosylation of mAbs and decreased basic charge variants in mAbs. For CS13‐1.00 cells, the mAb monomer increased with the addition of 40,000 Da DS at 0.3–1.0 g/L. Taken together, to maximize the beneficial effect of DS addition on mAb production, the optimal MW and concentration of DS should be determined for each specific rCHO cell line. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1113–1122, 2016     

Establishment of Alternative Culture Method for Spermatogonial Stem Cells Using Knockout Serum Replacement

Since spermatogonial stem cells (SSCs) are capable of both self-renewal and differentiation to daughter cells for subsequent spermatogenesis, the development of an efficient in vitro culture system is essential for studies related to spermatogenesis. Although the currently available system is serum-free and contains only chemically-defined components, it highly relies upon bovine serum albumin (BSA), a component with batch-to-batch quality variations similar to those of fetal bovine serum. Thus, we searched for an alternative BSA-free culture system that preserved the properties of SSCs. In this study, we utilized Knockout Serum Replacement (KSR) in the SSC culture medium, as a substitute for BSA. The results demonstrated that KSR supported the continuous growth of SSCs in vitro and the SSC activity in vivo without BSA, in a feeder-cell combination with mouse embryonic fibroblasts. The addition of BSA to KSR further facilitated cell cycle progression, whereas a transplantation assay revealed that the addition of BSA did not affect the number of SSCs in vivo. The combination of KSR with BSA also allowed the elimination of GFRA1 and FGF2, and the reduction of the GDNF concentration from 20 ng/ml to 5 ng/ml, while maintaining the growth rate and the expression of SSC markers. Furthermore, KSR was also useful with SSCs from non-DBA/2 strains, such as C57BL/6 and ICR. These results suggested that KSR is an effective substitute for BSA for long-term in vitro cultures of SSCs. Therefore, this method is practical for various studies related to SSCs, including spermatogenesis and germ stem cell biology.     

Embryoid body formation from embryonic and induced pluripotent stem cells: Benefits of bioreactors

Embryonic stem (ES) cells have the ability to differentiate into all germ layers, holding great promise not only for a model of early embryonic development but also for a robust cell source for cell-replacement therapies and for drug screening. Embryoid body (EB) formation from ES cells is a common method for producing different cell lineages for further applications. However, conventional techniques such as hanging drop or static suspension culture are either inherently incapable of large scale production or exhibit limited control over cell aggregation during EB formation and subsequent EB aggregation. For standardized mass EB production, a well defined scale-up platform is necessary. Recently, novel scenario methods of EB formation in hydrodynamic conditions created by bioreactor culture systems using stirred suspension systems (spinner flasks), rotating cell culture system and rotary orbital culture have allowed large-scale EB formation. Their use allows for continuous monitoring and control of the physical and chemical environment which is difficult to achieve by traditional methods. This review summarizes the current state of production of EBs derived from pluripotent cells in various culture systems. Furthermore, an overview of high quality EB formation strategies coupled with systems for in vitro differentiation into various cell types to be applied in cell replacement therapy is provided in this review. Recently, new insights in induced pluripotent stem (iPS) cell technology showed that differentiation and lineage commitment are not irreversible processes and this has opened new avenues in stem cell research. These cells are equivalent to ES cells in terms of both self-renewal and differentiation capacity. Hence, culture systems for expansion and differentiation of iPS cells can also apply methodologies developed with ES cells, although direct evidence of their use for iPS cells is still limited.     

Novel fluid shear‐based dissociation device for improved single cell dissociation of spheroids and cell aggregates

Biological industries commonly rely on bioreactor systems for the large‐scale production of cells. Cell aggregation, clumping, and spheroid morphology of certain suspension cells make their large‐scale culture challenging. Growing stem cells as spheroids is indispensable to retain their stemness, but large spheroids (>500 µm diameter) suffer from poor oxygen and nutrient diffusion, ultimately resulting in premature cell death in the centers of the spheroids. Despite this, most large‐scale bioprocesses do not have an efficient method for dissociating cells into single cells, but rely on costly enzymatic dissociation techniques. Therefore, we tested a proof‐of‐concept fluid shear‐based mechanical dissociator that was designed to dissociate stem cell spheroids and aggregates. Our prototype was able to dissociate cells while retaining high viability and low levels of apoptosis. The dissociator also did not impact long‐term cell growth or spheroid formation. Thus, the dissociator introduced here has the potential to replace traditional dissociation methods. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:293–298, 2018     

KSR1 is a functional protein kinase capable of serine autophosphorylation and direct phosphorylation of MEK1

The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway is a highly conserved signaling pathway that regulates diverse cellular processes including differentiation, proliferation, and survival. Kinase suppressor of Ras-1 (KSR1) binds each of the three ERK cascade components to facilitate pathway activation. Even though KSR1 contains a C-terminal kinase domain, evidence supporting the catalytic function of KSR1 remains controversial. In this study, we produced recombinant wild-type or kinase-inactive (D683A/D700A) KSR1 proteins in Escherichia coli to test the hypothesis that KSR1 is a functional protein kinase. Recombinant wild-type KSR1, but not recombinant kinase-inactive KSR1, underwent autophosphorylation on serine residue(s), phosphorylated myelin basic protein (MBP) as a generic substrate, and phosphorylated recombinant kinase-inactive MAPK/ERK kinase-1 (MEK1). Furthermore, FLAG immunoprecipitates from KSR1^−/− colon epithelial cells stably expressing FLAG-tagged wild-type KSR1 (+KSR1), but not vector (+vector) or FLAG-tagged kinase-inactive KSR1 (+D683A/D700A), were able to phosphorylate kinase-inactive MEK1. Since TNF activates the ERK pathway in colon epithelial cells, we tested the biological effects of KSR1 in the survival response downstream of TNF. We found that +vector and +D683A/D700A cells underwent apoptosis when treated with TNF, whereas +KSR1 cells were resistant. However, +KSR1 cells were sensitized to TNF-induced cell loss in the absence of MEK kinase activity. These data provide clear evidence that KSR1 is a functional protein kinase, MEK1 is an in vitro substrate of KSR1, and the catalytic activities of both proteins are required for eliciting cell survival responses downstream of TNF.     

A comparison of orbitally‐shaken and stirred‐tank bioreactors: pH modulation and bioreactor type affect CHO cell growth and protein glycosylation

Orbitally shaken bioreactors (OSRs) support the suspension cultivation of animal cells at volumetric scales up to 200 L and are a potential alternative to stirred‐tank bioreactors (STRs) due to their rapid and homogeneous mixing and high oxygen transfer rate. In this study, a Chinese hamster ovary cell line producing a recombinant antibody was cultivated in a 5 L OSR and a 3 L STR, both operated with or without pH control. Effects of bioreactor type and pH control on cell growth and metabolism and on recombinant protein production and glycosylation were determined. In pH‐controlled bioreactors, the glucose consumption and lactate production rates were higher relative to cultures grown in bioreactors without pH control. The cell density and viability were higher in the OSRs than in the STRs, either with or without pH control. Volumetric recombinant antibody yields were not affected by the process conditions, and a glycan analysis of the antibody by mass spectrometry did not reveal major process‐dependent differences in the galactosylation index. The results demonstrated that OSRs are suitable for recombinant protein production from suspension‐adapted animal cells. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1174–1180, 2016     

不同培养体系对绵羊类胚胎干细胞分离、传代的影响

以小鼠胚胎成纤维细胞(MEF)为饲养层, 研究了用Knockout血清替代品(Knockout serum replacement, KSR)代替胚胎干细胞(Embryonic stem cells, ES cell)培养液中的胎牛血清(FBS)和向含KSR的基础培养液中添加40%的小鼠ES细胞条件培养液(ES cell conditioned medium, ESCCM)对绵羊类ES细胞分离、克隆效率的影响。发现使用含FBS的基础培养液最多可以把绵羊类ES细胞传至3代, 而使用KSR和添加ESCCM能促进绵羊类ES细胞的分离和克隆, 所获得的类ES细胞分别可稳定传至第5和8代。同时对类ES细胞进行核型分析、AKP染色及体外分化能力检测, 证实所分离的类ES细胞符合ES细胞的主要特征。由此认为, 与FBS相比KSR更加适于绵羊类ES细胞的分离与培养; 而小鼠ES细胞在生长过程中可能分泌某些重要的细胞因子, 从而达到促进绵羊ES细胞增值的作用。     

Serum-free culture of murine primordial germ cells and embryonic germ cells

Fetal calf serum (FCS) has usually been used for culture of embryonic stem (ES) cell as a component of the culture medium. However, FCS contains undefined factors, which promote cell proliferation and occasionally stimulate differentiation of ES cells. Recently, a chemically-defined serum replacement, Knockout Serum Replacement (KSR), was developed to maintain ES cells in an undifferentiated state. In this experiment, we examined the effects of KSR on the growth and differentiation of primordial germ cells (PGCs) and embryonic germ (EG) cells. PGCs were collected 8.5 days postcoitum (dpc) from B6D2F1 (C57BL/6JxDBA/2J) female mice mated with B6D2F1 males. Most of the PGCs that were cultured in FCS-supplemented medium (FCS medium) had alkaline phosphatase (AP) activity and acquired a fibroblast cell shape. In contrast, PGCs in KSR-supplemented medium (KSR medium) proliferated, maintaining round and stem cell-like morphology. In addition, EG cells were established more easily from PGCs cultured in KSR medium than from PGCs cultured in FCS medium. The percentage of undifferentiated colonies of EG cells was significantly higher in KSR medium than in FCS medium. The germ line chimera was also produced from EG cells established in KSR medium. These results suggest that KSR can be used for sustaining an undifferentiated state of PGCs and EG cells in vitro.     

Detection of microbial contaminants in mammalian cell cultures using a new fluorescence-based staining method

Aims: Microbial contamination of cell culture production processes is a current concern for biopharmaceutical industries. Traditional testing methods require several days to detect contamination and may advantageously be replaced by a rapid detection method. We developed a new method combining membrane filtration to microcolonies fluorescence staining method (MFSM) and compared it to epifluorescence microscopy. Methods and Results: Both methods were used to detect bacteria in CHO cells cultures. The epifluorescence microscopy showed to be limited by filterability, media interference and nonrobustness issues, whereas MFSM enabled consistent detection of Bacillus cereus, Staphylococcus epidermidis and Propionibacterium acnes after, respectively, 8, 9 and 48 h of incubation. Thanks to the nondestructive feature of the MFSM, stained membranes could be reincubated on culture media to yield visible colonies used for identification. Conclusions: The new method described in this study showed its ability to detect microbial contaminants in cell culture samples with time‐to‐results from 2–5 times shorter than the traditional testing method. Significance and Impact of the Study: The MFSM can be used as monitoring tool for cell cultures to significantly shorten detection times of microbial contamination, while preserving the ability to identify the contaminants and their viability.     

Characterization of functionally active interleukin‐18/eGFP fusion protein expression during cell cycle phases in recombinant chicken DF1 Cells

The dependence of foreign gene expression on cell cycle phases in mammalian cells has been described. In this study, a DF1/chIL‐18a cell line that stably expresses the fusion protein chIL‐18 was constructed and the enhanced green fluorescence protein connected through a (G₄S)₃ linker sequence investigated the relationship between cell cycle phases and fusion protein production. DF1/chIL‐18a cells (1 × 10⁵) were inoculated in 60‐mm culture dishes containing 5 mL of media to achieve 50%–60% confluence and were cultured in the presence of the cycle‐specific inhibitors 10058‐F4, aphidicolin, and colchicine for 24 and 48 h. The percentage of cell density and mean fluorescence intensity in each cell cycle phase were assessed using flow cytometry. The inhibitors effectively arrested cell growth. The fusion protein production rate was higher in the S phase than in the G0/G1 and G2/M phases. When cell cycle progression was blocked in the G0/G1, S, and G2/M phases by the addition of 10058‐F4, aphidicolin, and colchicine, respectively, the aphidicolin‐induced single cells showed higher fusion protein levels than did the 10058‐F4‐ or colchicine‐induced phase cells and the uninduced control cells. Although the cells did not proliferate after the drug additions, the amount of total fusion protein accumulated in aphidicolin‐treated cells was similar to that in the untreated cultures. Fusion protein is biologically active because it induces IFN‐γ production in splenocyte cultures of chicken. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:581–591, 2016     

A scale‐down mimic for mapping the process performance of centrifugation,depth and sterile filtration

In the production of biopharmaceuticals disk‐stack centrifugation is widely used as a harvest step for the removal of cells and cellular debris. Depth filters followed by sterile filters are often then employed to remove residual solids remaining in the centrate. Process development of centrifugation is usually conducted at pilot‐scale so as to mimic the commercial scale equipment but this method requires large quantities of cell culture and significant levels of effort for successful characterization. A scale‐down approach based upon the use of a shear device and a bench‐top centrifuge has been extended in this work towards a preparative methodology that successfully predicts the performance of the continuous centrifuge and polishing filters. The use of this methodology allows the effects of cell culture conditions and large‐scale centrifugal process parameters on subsequent filtration performance to be assessed at an early stage of process development where material availability is limited. Biotechnol. Bioeng. 2016;113: 1934–1941. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Controlling the time evolution of mAb N‐linked glycosylation,Part I: Microbioreactor experiments

N‐linked glycosylation is of key importance for the efficacy of many biotherapeutic proteins such as monoclonal antibodies (mAbs). Media components and cell culture conditions have been shown to significantly affect N‐linked glycosylation during the production of glycoproteins using mammalian cell fed‐batch cultures. These parameters inevitably change in modern industrial processes with concentrated feed additions and cell densities beyond 2 × 10⁷ cells/mL. In order to control the time‐dependent changes of protein glycosylation, an automated microbioreactor system was used to investigate the effects of culture pH, ammonia, galactose, and manganese chloride supplementation on nucleotide sugars as well as mAb N‐linked glycosylation in a time‐dependent way. Two different strategies comprising of a single shift of culture conditions as well as multiple media supplementations along the culture duration were applied to obtain changing and constant glycosylation profiles. The different feeding approaches enabled constant glycosylation patterns throughout the entire culture duration at different levels. By modulating the time evolution of the mAb glycan pattern, not only the endpoint but also the ratios between different glycosylation structures could be modified. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1123–1134, 2016     

SHORT‐TERM EFFECTS OF ACETATE AND MICROAEROBIC CONDITIONS ON PHOTOSYNTHESIS AND RESPIRATION IN CHLORELLA SOROKINIANA GXNN 01 (CHLOROPHYTA)1

The culture of microalgae using organic carbon sources decreases the cost of operation in closed systems. The effect of carbon sources on microalgae is thus an interesting problem in not only theoretical research but also practical production. The short‐term effects of acetate and microaerobic conditions on the growth, photosynthesis, and respiration of the green microalga Chlorella sorokiniana I. Shihira & R.W. Krauss GXNN 01 were described after acetate addition to autotrophic cultures. As the acetate concentration increased, cells needed a longer lag phase to grow, and 243.8 mM acetate completely inhibited growth. Acetate addition induced an immediate response in photosynthesis and respiration. The activity of PS II and PS I were impaired and declined with different rates, and then recovered compared with autotrophic cells. Carbonic anhydrase and Rubisco activities were also inhibited at the beginning, and respiration was increased. We propose that ATP consumption for acetate assimilation results in surplus NADPH, and then accumulated reducing power over‐reduces inter‐photosystem components and raises the transthylakoid proton gradient, which redistributes energy between PS I and PS II, and leads to a decrease in the PS II/PS I ratio and O₂ evolution. An apparent cyclic electron flow was also observed, which may be mainly mediated by NAD(P)H dehydrogenase‐dependent pathway since NADPH was in excess. These observations pointed to an acclimation process after acetate addition, and suggested the interaction between photosynthesis and respiration involving ATP and reducing power.     

Serum‐free scaled up expansion and differentiation of murine embryonic stem cells to osteoblasts in suspension bioreactors

The use of embryonic stem cell (ESC) derived cells has emerged as a potential alternative treatment for a number of degenerative diseases, including musculoskeletal diseases. Conventional ESC culturing methods use fetal bovine serum (FBS) as a major supplemental component of culture media, which is undesirable for clinical applications. These cultures are usually performed in small‐scale static vessels (gelatin‐coated dishes), which limit the number of cells that can be generated. It is essential to develop effective, reproducible protocols for efficient scalable production of ESC‐derived cells. Here we present serum‐free bioreactor protocols for (1) expansion and (2) differentiation of embryonic stem cells to osteoblasts. Cultivation of mESCs in serum‐free media, supplemented with 15% knockout serum replacement (KSR) resulted in a 27.1‐ and 48.6‐fold expansion in static culture and suspension respectively by day 5 of culture. Further induction to osteoblasts with a differentiation cocktail was verified by up‐regulation of osterix and osteocalcin. Mineralization was also enhanced, as indicated by an increase in the calcium deposition by osteogenic cells by day 28. These results will serve as the basis for developing protocols with human ESCs as a new treatment alternative for musculoskeletal diseases. Biotechnol. Bioeng. 2010;106: 829–840. © 2010 Wiley Periodicals, Inc.     

Evaluating the toxicity of bDtBPP on CHO‐K1 cells for testing of single‐use bioprocessing systems considering media selection,cell culture volume,mixing, and exposure duration

Single‐use bioprocessing bags are gaining popularity due to ease of use, lower risk of contamination, and ease of process scale‐up. Bis(2,4‐di‐tert‐butylphenyl)phosphate (bDtBPP), a degradant of tris(2,4‐di‐tert‐butylphenyl)phosphite, marketed as Irgafos 168®, which is an antioxidant stabilizer added to resins, has been identified as a potentially toxic leachate which may impact the performance of single‐use, multilayer bioprocessing bags. In this study, the toxicity of bDtBPP was tested on CHO‐K1 cells grown as adherent or suspended cells. The EC50 (effective concentration to cause 50% cell death) for adherent cells was found to be one order of magnitude higher than that for suspended CHO‐K1 cells. While CHO‐K1 cells had good cell viability when exposed to moderate concentrations of bDtBPP, the degradant was shown to impact the viable cell density (VCD) at much lower concentrations. Hence, in developing an industry‐standard assay for testing the cytotoxicity of leachates, suspended cells (as commonly used in the bioprocessing industry) would likely be most sensitive, particularly when reporting EC50 values based on VCD. The effects of mixing, cell culture volume, and exposure duration were also evaluated for suspended CHO‐K1 cells. It was found that the sensitivity of cell culture to leachates from single‐use plastic bags was enhanced for suspended cells cultured for longer exposure times and when the cells were subjected to continuous agitation, both of which are important considerations in the production of biopharmaceuticals. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1318–1323, 2016     

Testing plasmid stability of Escherichia coli using the Continuously Operated Shaken BIOreactor System

Plasmids are common vectors to genetically manipulate Escherichia coli or other microorganisms. They are easy to use and considerable experience has accumulated on their application in heterologous protein production. However, plasmids can be lost during cell growth, if no selection pressure like, e.g., antibiotics is used, hampering the production of the desired protein and endangering the economic success of a biotechnological production process. Thus, in this study the Continuously Operated Shaken BIOreactor System (COSBIOS) is applied as a tool for fast parallel testing of strain stability and operation conditions and to evaluate measures to counter such plasmid loss. In specific, by applying various ampicillin concentrations, the lowest effective ampicillin dosage is investigated to secure plasmid stability while lowering adverse ecological effects. A significant difference was found in the growth rates of plasmid‐bearing and plasmid‐free cells. The undesired plasmid‐free cells grew 30% faster than the desired plasmid‐bearing cells. During the testing of plasmid stability without antibiotics, the population fraction of plasmid‐bearing cells rapidly decreased in continuous culture to zero within the first 48 h. An initial single dosage of ampicillin did not prevent plasmid loss. By contrast, a continuous application of a low dosage of 10 µg/mL ampicillin in the feed medium maintained plasmid stability in the culture. Consequently, the COSBIOS is an apt reactor system for measuring plasmid stability and evaluating methods to enhance this stability. Hence, decreased production of heterologous protein can be prevented. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1418–1425, 2016     

Patterns of protein expression upon adding sugar and elicitor to the cell culture of Eschscholtzia californica

The optimal timing of elicitation was determined for the production of benzophenanthridine alkaloids (BPAs) by Eschscholtzia californica cell culture. Upon elicitation, 7-day old cells produced more alkaloids than 14-day old cells (5.1 times for sanguinarine and 2.7 times for dihydrosanguinarine). We presumed that these alkaloids are growth-rate-associated secondary metabolites in E. californica cell culture. Although the specific productivity of alkaloids were higher in 7-day old culture, the total cell mass of 7-day old culture was about half that of 14-day old culture. In order to increase the overall productivity, sucrose was added to the 14-day old culture before the addition of elicitor. By this way, cells in the stationary phase (14-day old culture) could be switched to the cells in the logarithmic growth phase (similar to 7-day old culture). Total production of alkaloids was increased by adding sucrose; especially the production of sanguinarine was increased as high as 5.7 times of the control. To find out the protein level changed by the elicitation, proteins extracted from whole cell were separated by using two-dimensional gel electrophoresis. The patterns of the gels were different and little correlation among the proteins could be observed. And Western blotting was employed to check the expression level of selected five enzymes, these enzymes believed to be involved in BPAs production, resulting in up-regulated with elicitor addition.     

High density continuous production of murine pluripotent cells in an acoustic perfused bioreactor at different oxygen concentrations

Strategies for the production of pluripotent stem cells (PSCs) rely on serially dissociated adherent or aggregate‐based culture, consequently limiting robust scale‐up of cell production, on‐line control and optimization of culture conditions. We recently developed a method that enables continuous (non‐serially dissociated) suspension culture‐mediated reprogramming to pluripotency. Herein, we use this method to demonstrate the scalable production of PSCs and early derivatives using acoustic filter technology to enable continuous oxygen‐controlled perfusion culture. Cell densities of greater than 1 × 10⁷ cells/mL were achieved after 7 days of expansion at a specific growth rate (µ) of 0.61 ± 0.1 day^?1 with a perfusion rate (D) of 5.0 day^?1. A twofold increase in maximum cell density (to greater than 2.5 × 10⁷ cells/mL) was achieved when the medium dissolved oxygen was reduced (5% DO). Cell densities and viabilities >80% were maintained for extended production periods during which maintenance of pluripotency was confirmed by stable expression of pluripotency factors (SSEA‐1 and Nanog), as well as the capacity to differentiate into all three germ layers. This work establishes a versatile biotechnological platform for the production of pluripotent cells and derivatives in an integrated, scalable and intensified stirred suspension culture. Biotechnol. Bioeng. 2013; 110: 648–655. © 2012 Wiley Periodicals, Inc.     

Aggregation of bovine anterior cruciate ligament fibroblasts or marrow stromal cells promotes aggrecan production

The development of a tissue‐engineered alternative for current ligament grafts requires the creation of a fibrocartilaginous interface between the engineered ligament midsubstance and bone tissue. Therefore, the focus of this study was to examine the potential for cartilaginous extracellular matrix (ECM) formation by altering culture parameters for bovine anterior cruciate ligament (ACL) fibroblasts and marrow stromal cells (MSCs). Specifically, cells were cultured without chondrogenic media supplements on aggrecan‐coated surfaces, tissue culture‐treated control surfaces, and nonadhesive surfaces that promoted cell aggregation, and examined over 14 days. Aggrecan‐coated surfaces promoted the aggregation of ACL fibroblasts and MSCs within 24 h after seeding. Aggrecan gene expression was significantly upregulated in cell aggregates, regardless of how cell clustering was induced, with as much as 10.9 ± 1.2‐fold upregulation in ACL fibroblasts and 9.7 ± 1.1‐fold in MSCs after 3 days, compared to control surfaces. Dimethylmethylene blue (DMMB) results and immunostaining verified the presence of aggrecan in ACL fibroblast and MSC aggregates throughout the culture period. Results indicate that ACL fibroblasts retained the ability to alter their gene expression and produce aggrecan, though MSCs, in general, had a more consistent response to aggregation. These findings support the use of aggregate‐inducing materials to encourage production of aggrecan and suggest that altering the degree of clustering could produce a range of phenotypes from a single cell source. As such, this represents a first step which may inform future approaches to producing tissue‐engineered ligament grafts. Biotechnol. Bioeng. 2011; 108:151–162. © 2010 Wiley Periodicals, Inc.     

TNT‐Induced Phagocytosis: Tunneling Nanotubes Mediate the Transfer of Pro‐Phagocytic Signals From Apoptotic to Viable Cells

The exposure of phosphatidylserine (PS) on the surface membrane of apoptotic cells triggers the recruitment of phagocytic receptors and subsequently results in uptake by phagocytes. Here we describe how apoptotic cells can use intercellular membrane nanotubes to transfer exposed PS to neighboring viable cells, and thus deposit an “eat‐me” tag on the viable cells. Tunneling nanotubes (TNTs) connected UV‐treated apoptotic rat pheochromocytoma PC12 cells with neighboring untreated cells. These TNTs were composed of PS‐exposed plasma membrane and facilitated the transfer of the membrane from apoptotic to viable cells. Other pro‐phagocytic signals, such as oxidized phospholipids and calreticulin, were also transferred to viable cells. In addition, anti‐phagocytic signal CD47 presenting on the plasma membrane of viable cells was masked by the transferred PS‐membrane. Confocal imaging revealed an increase of phagocytosis of viable PC12 cells by murine RAW264.7 macrophages when the viable PC12 cells were cocultured with UV‐treated PC12 cells. Treatment with 50 nM cytochalasin D would abolish TNTs and correspondingly inhibit this phagocytosis of the viable cells. Our study indicates that exposed‐PS membrane is delivered from apoptotic to viable cells through TNTs. This transferred membrane may act as a pro‐phagocytic signal for macrophages to induce phagocytosis of viable cells in a situation where they are in the vicinity of apoptotic cells. J. Cell. Physiol. 232: 2271–2279, 2017. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals Inc.