Presented here are techniques developed to culture and analyze three-dimensional (3-D) adipose-like tissues as a means to bridge the gap between current limitations in culturing preadipocytes (PAs) and that of providing clinically relevant volumes of adipose tissue useful for soft tissue engineering strategies in reconstructive surgery. Pilot studies were performed to determine techniques to visualize and analyze 3-D PA-like tissues as well as to develop successful strategies to culture 3T3-L1 cells in a high aspect ratio vessel rotating-wall bioreactor both with and without microcarriers. Next, a series of cultures were accessed to verify these techniques as well as to compare the culture of the cells with and without microcarriers. Finally, a perfused rotating-wall bioreactor was used to further investigate the nature of the aggregates or tissues being generated. The aggregates that formed in the perfused system were analyzed via histology and in vivo animal studies. PA-like tissues as large as 4–5 mm in diameter without microcarriers that were capable of lipid-loading and composed of viable cells were achieved. We have successfully demonstrated that large tissue aggregates can be grown in bioreactor culture systems.

The differentiation of mouse embryonic stem (ES) cells can be induced in vitro after leukemia inhibitory factor (LIF) withdrawal and further enhanced by the formation of “embryoid body” (EB) aggregates. This strategy is being used in order to optimize differentiation protocols that would result in functional cells for experimental cell replacement therapies. However, this study presents the possibility for residual undifferentiated cells to survive after standard in vitro procedures. Mouse ES cells were stably transfected with the enhanced green fluorescent protein (EGFP), under the control of the Oct4 promoter, a transcription factor that is expressed in undifferentiated ES cells but down-regulated on differentiation. Residual fluorescent cells were isolated from EBs that were cultured in standard conditions in absence of LIF. These residual cells displayed recurrent gain of chromosomes 8 and 9. Residual fluorescent cells, further expanded in absence of LIF and cultured as EBs, still displayed a significant Oct4 expression in comparison with parental transfected ES cells. Consequently, these residual cells have an intrinsic resistance to differentiate. The behavior of these cells, observed in vitro, can be overcome in vivo, as they were able to induce teratomas in subcutaneously injected nude mice. Residual undifferentiated cells displayed slight levels of VASA and DAZL expression. These results demonstrate that mouse ES cells cultured in vitro, in standard conditions, can spontaneously acquire recurrent karyotypical changes that may promote an undifferentiated stage, being selected in standard culture conditions in vitro.

Low oxygen tension (hypoxia) has been implicated in proliferation of vascular smooth muscle cells (SMCs) of the lung. Tissue hypoxia also occurs in the obstructed bladder. The extracellular-regulated kinase mitogen-activated protein kinase 1/2 (Erk1/2) pathway is induced in many cell types during hypoxia. We examined whether hypoxia (3% O₂), compared with normoxia (21% O₂), induces proliferation responses and activation of the Erk1/2 pathways in primary rat bladder smooth muscle cells (BSMCs). We show that hypoxia induces proliferation of BSMCs at 18 h and, although reduced at 22 h, still remained above normoxic levels. Hypoxia induced a strikingly transient activation of Erk1/2 that lasted only 10–30 min. However, inhibition of the transient Erk1/2 activity with a specific mitogen-activated protein kinase kinase 1 (MEK-1) inhibitor PD 98059 prevented subsequent hypoxia-induced proliferation at 18 h. Interestingly, inhibition of general matrix metalloproteinase (MMP) activity, using either doxycycline or GM 6001, prevented both transient Erk1/2 activity and subsequent proliferation in response to hypoxia. Furthermore, MMP-7 (matrilysin) is activated in the conditioned medium (CM) of BSMCs at 10–20 min of hypoxia. In addition, MMP-7 was also transcriptionally induced at 6 h of hypoxia in an Erk1/2-dependent manner. Moreover, transient Erk1/2 activation and BSMC proliferation were both dependent on epidermal growth factor receptor (EGFR/HER1) but not neu receptor (HER2/ERB2) autophosphorylation. We conclude that hypoxia leads to Erk1/2 activation, which appears to modulate BSMC proliferation through MMP-7-and EGFR-mediated mechanisms.

To examine whether a neuronal cell suspension can be held in vitro for a relatively short period without compromising survival rates and functionality, we have set up an experimental protocol planning 24 h of suspension culture in a rotary wall vessel (RWV) bioreactor before plating in a conventional adherent system. Apoptosis measurement and activated caspase-8, -9, and -3 detection have demonstrated that survey of the cells was not affected. The activity of major antioxidant enzymes (AOE), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT), was significantly decreased in RWV-maintained cells. A significant decrease of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) is coupled with a level of activated nuclear factor-κB (NF-κB) protein significantly lower in RVW cells than in the control. On the contrary, the level of IL-6 expression did not change between the test and the control. A significant up-regulation of growth-associated protein-43 (GAP-43), peroxisome proliferator-activated receptor-β/δ (PPARβ/δ), and acyl-CoA synthetase 2 (ACS2) in RWV cells has been detected. We provide the evidence that primary neuronal cells, at an early stage of development, can be maintained in a suspension condition before adherent plating. This experimental environment does not induce detrimental effects but may have an activator role, leading cells to development and maturation in a tridimensional state.

We have established a means for prolonged survival of primary cell cultures and establishment of continuous cell lines without genetic manipulations. Primary cultures of granulosa cells degenerate rapidly in vitro by a spontaneous onset of apoptotic cell death. Earlier attempts to circumvent this limitation have included transformation with oncogenes, spontaneous immortalization of primary cultures, and chemical carcinogenesis. We have found that addition of a complex of growth-promoting compounds, carrier proteins, and factors isolated from porcine follicular fluid to standard culture medium allows, reproducibly, the establishment of continuous porcine primary granulosa cell lines with genetic stability. This same supplement allows the prolonged survival of primary cell cultures derived from adult rat ovaries. The rat ovary primary cultures consisted of mixed phenotypes, including epithelial, neuron-like, and mesenchymal cell types. Numerous cells stain positive for alkaline phosphatase in these cultures. Other primary cell lines were established from embryonic rat liver and from adult rat lungs, using the same supplement. The survival effect is reversible because cells degenerate when the supplement is removed. Therefore, the cell lines have neither acquired properties of a tumor cell line nor have they been immortalized by a virus infection. We expect that our approach will open the door to prolonged survival of other primary cell types.

Three insect cell lines, IPLB-LdFB and IPLB-LdEIta from gypsy moth fat body and embryos and UFL-AG-286 from velvetbean caterpillar embryos, have been concurrently maintained for 1 to 12 yr on two media formulations, modified TC-100 containing 9% fetal bovine serum and Ex-cell 400, a commercial serum-free medium (SFM). Cells grown in each medium were tested for susceptibility to and productivity of various multiply embedded nucleopolyhedroviruses. The three lines chosen for these experiments fall into three categories of relative growth in SFM versus TC-100: LdFB cells grew similarly in each medium, LdEIta grew better in Ex-Cell than in TC-100, and AG-286 grew better in TC-100 than in Ex-Cell. The susceptibility of cells to infection also varies, although without any apparent correlation to which medium was best for supporting growth. Endpoint assays suggested that LdFB cells grown in serum-containing medium are more susceptible to virus infection than their SFM counterparts, while the opposite is true for LdEIta cells. Production of virus, based on numbers of occlusion bodies, showed fewer differences with only AcMNPV production with AG-286 in TC-100 being statistically higher than production of the same virus in Ex-cell 400. These studies suggest that long-term passage in alternative media may impact the ability of cells to support virus infection and replication, but the effects on each cell line and virus system need to be determined.

T lymphocytes cultured under the low-shear stress environment of modeled microgravity demonstrate an inhibition of activation in response to T-cell receptor (TCR)–mediated signaling. Modeled microgravity culture-induced inhibition mimics the inhibition observed during spaceflight. This work investigates the molecular signaling events of interleukin 2 transcription activation in modeled microgravity as generated with clinorotation. Under normal conditions, NFAT (nuclear factor of activated T cells) is dephosphorylated and activated with sustained calcium (Ca ) influx and calcineurin activity, whereas AP-1 is activated by protein kinase C (PKC) and Ras-mediated pathways. Purified human T lymphocytes are shown to exhibit differential inhibition of transcription factor activation in modeled microgravity. Activation of AP-1 is blocked with clinorotation, whereas NFAT dephosphorylation occurs. This work supports the theory that modeled microgravity differentially blocks the activation of distinct signaling mechanism.

In recent years, a great variety of different matrix systems for the cultivation of chondrocytes have been developed. Although some of these scaffolds show promising experimental results in vitro, the potential clinical value remains unclear. In this comparative study, we propagated human articular chondrocytes precultivated in monolayer culture on six different scaffolds (collagen gels, membranes and sponges) under standardized in vitro conditions. Mechanical properties of the matrix systems were not improved significantly by cultivation of human chondrocytes under the given in vitro conditions. The gel systems (CaReS, Ars Artho, Germany and Atelocollagen, Koken, Japan) showed a homogeneous cell distribution; chondrocytes propagated on Chondro-Gide (Geistlich Biomaterials, Switzerland) and Integra membranes (Integra, USA) were building multilayers. Only few cells penetrated the two Atelocollagen honeycomb sponges (Koken, Japan). During cultivation, chondrocytes propagated on all systems showed a partial morphological redifferentiation, which was best with regard to the gel systems. In general, only small amounts of collagen type-II protein could be detected in the pericellular region and chondrocytes failed to build a territorial matrix. During the first two weeks of cultivation, the two gel systems showed a significantly higher collagen type-II gene expression and a lower collagen type-I gene expression than the other investigated matrix systems. Although collagen gels seem to be superior when dealing with deep cartilage defects, membrane systems might rather be useful in improving conventional autologous chondrocyte transplantation or in combination with gel systems.