The success of insect cell culture is demonstrated by reports of over 500 established cell lines. While established procedures that can be used for developing new cell lines exist, these usually require some fine-tuning for various tissue sources. This paper attempts to depict some of the variations that can be applied.

Creation of a shrimp cell line has been an elusive goal. This failure may be due to the composition of the cell culture medium, which may be inadequate to support primary cultured cells. Shrimp hemolymph should contain the nutritional components needed to support cell growth and division. We report here the comprehensive biochemical analysis of hemolymph from the blue shrimp, Penaeus stylirostris (Litopenaeus stylirostris) (see Holthuis, L. B. Shrimps and prawns of the world, in: FAO species catalog. Vol. 1. Rome: Food and Agriculture Organization of the United Nations; 1980), for free amino acids (FAAs), carbohydrates, electrolytes, metals, pH, and osmolality. Levels of hemolymph components were compared to 2×L-15 with 20% fetal bovine serum, a commonly used culture medium for crustacean cells. The FAAs, taurine and proline, and the metals, strontium and zinc, were significantly higher in hemolymph than in the 2×L-15 medium. In contrast, other FAAs were up to 50 times higher in the 2×L-15 medium than in the hemolymph. To mimic more closely the hemolymph composition, we created two new media based on either the 0.2×L-15 or the M199 medium. We compared the microscopic appearance of cells cultured in these media and evaluated deoxyribonucleic acid (DNA) and protein synthesis by ³H-thymidine uptake and ³⁵S-methionine uptake assays. The ovary cells of P. stylirostris cultured in either of the new media formed monolayers, while the cells cultured in 2×L-15 medium did not. Despite these differences, there was no evidence of sustained DNA or protein synthesis with any of the media. Future studies to establish a shrimp cell line should focus on analysis of the cell cycle and on overcoming the molecular blocks to cell division.

A highly nutritive culture medium (MGM-464) was developed for insect cell primary culture. The new medium consists of 6 inorganic salts, 4 organic acids, 21 amino acids, 3 sugars, 10 vitamins, and 8 other chemicals, including natural substances. The complete medium was generated by adding 20 ml fetal bovine serum to 100 ml MGM-464. The detail of the composition of the medium is given in a table, and the protocol to prepare the medium is described in the text. Among the 15 kinds of cultures made with MGM-464, embryonic cells from a walking stick and ovarian cells from the common white were subcultured more than 70 times, and embryonic cells of a chrysomelid beetle were subcultured more than 15 times. Other cultures could not be subcultured. However, embryonic cells from the commercial silkworm and a cockroach, ovarial cells from the commercial silkworm and a sphingid moth, nervous cells from the commercial silkworm and two sphingid moths, and cells from the dorsal vessel plus surrounding tissue of the commercial silkworm survived for several mo. The cells from the honeybee embryos, aphid embryos, and planthopper embryos were rather short-lived, and deteriorated after about 1 mo.

The Manduca sexta (L.) [Lepidoptera: Sphingidae] and Heliothis virescens (F.) [Lepidoptera: Noctuidae] midguts consist of a pseudostratified epithelium surrounded by striated muscle and tracheae. This epithelium contains goblet, columnar, and basal stem cells. The stem cells are critically important in that they are capable of massive proliferation and differentiation. This growth results in a fourfold enlargement of the midgut at each larval molt. The stem cells are also responsible for limited cell replacement during repair. While the characteristics of the stem cell population vary over the course of an instar, stem cells collected early in an instar and those collected late can start in vitro cultures. Cultures of larval stem, goblet, and columnar cells survive in vitro for several mo through proliferation and differentiation of the stem cells. One of the two polypeptide differentiation factors which have been identified and characterized from the culture medium has now been shown to be present in midgut in vivo. Thus the ability to examine lepidopteran midgut stem cell growth in vitro and in vivo is proving to be effective in determining the basic features of stem cell action and regulation.

The number of insect midgut cells is maintained homeostatically in vivo and in vitro. However, during starvation, the midgut shrinks and the rate of cell replacement appears to be suppressed. When they undergo metamorphosis, the internal organs of insects are drastically remodeled by cell proliferation, differentiation, and apoptotic processes, and the net number of cells usually increases. An extract of 1650 midguts of Periplaneta americana was fractionated by high-performance liquid chromatography (HPLC) to obtain the peptides that regulate these processes. The HPLC fractions were tested for myotropic activity in the foregut and for effects on cell proliferation or loss in primary cultures of larval Heliothis virescens midgut cells and in a cell line derived from the last-instar larval fat body of Mamestra brassicae. Some fractions stimulated midgut stem cell proliferation and differentiation, while others caused loss of differentiated columnar and goblet cells. Other fractions stimulated cell proliferation in the larval fat body cells.

Differentiated cells in the insect midgut depend on stem cells for renewal. We have immunologically identified Integrin β₁, a promotor of cell–cell adhesion that also induces signals mediating proliferation, differentiation, and apoptosis on the surfaces of cultured Heliothis virescens midgut cells; clusters of immunostained integrin β₁–like material, indicative of activated integrin, were detected on aggregating midgut columnar cells. Growth factor–like peptides (midgut differentiation factors 1 and 2 [MDF1 and MDF2]), isolated from conditioned medium containing Manduca sexta midgut cells, may be representative of endogenous midgut signaling molecules. Exposing the cultured midgut cells to Bacillus thuringiensis (Bt) toxin caused large numbers of mature differentiated cells to die, but the massive cell death simultaneously induced a 150–200% increase in the numbers of midgut stem and differentiating cells. However, after the toxin was washed out, the proportions of cell types returned to near-control levels within 2 d, indicating endogenous control of cell-population dynamics. MDF1 was detected immunologically in larger numbers of Bt-treated columnar cells than controls, confirming its role in inducing the differentiation of rapidly produced stem cells. However, other insect midgut factors regulating increased proliferation, differentiation, as well as inhibition of proliferation and adjustment of the ratio of cell types, remain to be discovered.

Midgut epithelial cells were isolated from fifth-instar Pseudaletia unipuncta larvae by collagenase treatment of midgut tissue, and cultured in TNM-FH medium. Long-term continuous culture and maintenance of midgut cells were achieved with P. unipuncta armyworm intestinal cells. Several cells lines were obtained from these P. unipuncta primary cultures, and they have been subcultured and maintained for over 24 mo. The three major midgut cell types were present in the cultures, including stem (regenerative), columnar, and goblet cells. In vitro morphogenesis and differentiation of columnar and goblet cells from stem cells were observed. There appeared to be a cycle of cell death of goblet and columnar cells followed by their replacement from stem cells every 7–8 wk. After approximately six passages, the cell density in T-flasks appeared to be somewhat constant, reaching 10³–10⁴ cells per milliliter of medium. The columnar cells are round to rectangular in shape and possess a brush border, while the goblet cells have a classic flask-like shape with a central cavity. Peritrophic membrane-like secretions were observed in all the culture flasks. Infection of these cells with multiply embedded nucleopolyhedrovirus was confirmed, and we conclude that these midgut cells can be used as an in vitro model system to study early events in baculovirus infection.

Cell lines from selected lepidopteran species were established for the overall purpose of use in baculovirus production. A total of 36 new cell lines from 10 lepidopteran species were generated, including cell lines from a pyralid, the European corn borer, Ostrinia nubilalis, a plutellid, the diamondback moth, Plutella xylostella, as well as eight noctuids: the black cutworm, Agrotis ipsilon, the celery looper, Anagrapha falcifera, the velvetbean caterpillar, Anticarsia gemmatalis, the corn earworm, Helicoverpa zea, the tobacco budworm, Heliothis virescens, the beet armyworm, Spodoptera exigua, the fall armyworm, Spodoptera frugiperda, and the cabbage looper, Trichoplusia ni. Tissues used for cell line establishment included fat bodies, ovaries, testes, or whole embryos/larvae/pupae. All the cell lines were subcultured numerous times, characterized by isoenzyme analysis and/or deoxyribonucleic acid amplification fingerprinting using polymerase chain reaction, and stored in liquid nitrogen. Many of the cell lines were adapted to grow in serum-free medium, with cell lines from A. ipsilon and H. virescens being adapted to suspension culture using shaker flasks. The potential use for these cell lines in baculovirus production is discussed.

One key to the in vitro mass production of baculoviruses is the development of insect cell lines capable of producing high levels of extracellular virus (ECV) and/or occlusion bodies (OBs). For this study, 34 newly established cell lines from 10 lepidopteran species were screened for their ability to produce ECV and OBs from a variety of baculoviruses. The selected baculoviruses included: the alfalfa looper virus (AcMNPV); the celery looper virus (AfMNPV); the velvetbean caterpillar virus (AgMNPV), the bollworm virus (HzSNPV), the diamondback moth virus (PxMNPV), and the beet armyworm virus (SeMNPV). ECV titers were determined using TCID₅₀ assays (50% tissue culture infectivity dose), with the presence or absence of OBs being noted. For AcMNPV, 28 new cell lines were tested, with eight producing AcMNPV ECV titers of 1.1–47.3 × 10⁶ TCID₅₀/ml and 11 producing OBs. For AgMNPV, six new cell lines were tested, with all producing AgMNPV ECV titers of 3.5–62.3 × 10⁶ TCID₅₀/ml and generating OBs. For HzSNPV, four new cell lines were tested with three lines producing HzSNPV ECV titers of 1.4–5.0 × 10⁶ TCID₅₀/ml, but none generating OBs. For PxMNPV, 10 new cell lines were tested with seven generating PxMNPV ECV titers of 4.7–232.6 × 10⁶ TCID₅₀/ml and eight producing OBs. Lastly, using qualitative or semiquantitative methods, homologous cell lines were tested for AfMNPV and SeMNPV production, all of which produced OBs. Overall, many of the cell lines tested were found to produce OBs and generate moderate to high levels of ECVs of one or more baculoviruses.

This study was undertaken to examine the influence of time and volume of collagen overlay, type of media, and media additives on taurocholate (TC) accumulation and biliary excretion in hepatocytes cultured in a collagen-sandwich configuration. Hepatocytes were isolated from male Wistar rats by in situ perfusion with collagenase, seeded onto collagen-coated 60-mm dishes, overlaid with gelled collagen, and cultured for 4 d. Experiments to examine the influence of time and volume of collagen overlay were conducted in Dulbecco's modified Eagle's medium (DMEM) 1.0 μM dexamethasone (DEX) 5% fetal bovine serum (FBS). Hepatocytes were overlaid at 0 h with 0.1 or 0.2 ml collagen, or at 24 h with 0.1 or 0.2 ml collagen. The influence of media type and additives was examined in hepatocytes overlaid at 0 h with 0.2 ml collagen and incubated in DMEM 0.1 μM DEX, DMEM 0.1 μM DEX 5% FBS, Williams' medium E 0.1 μM DEX 1% ITS^™ , DMEM 1.0 μM DEX, DMEM 1.0 μM DEX 5% FBS, or modified Chee's medium (MCM) 0.1 μM DEX 1% ITS^™ . [³H] TC accumulation by hepatocytes in Hank's balanced salt solution (HBSS) and Ca² -free HBSS was measured, and the biliary-excretion index (BEI: percentage of accumulated TC localized in the canalicular compartment) was calculated. Light microscopy and carboxydichlorofluorescein fluorescence were employed to examine the cellular and canalicular morphologies. The volume of collagen used for both the substratum and the overlay did not affect TC accumulation or biliary excretion. The BEI tended to be higher in cells overlaid at 24 h (BEI = 0.649 [0.1 ml collagen]; BEI = 0.659 [0.2 ml collagen]) compared with those overlaid at 0 h after seeding (BEI = 0.538 [0.1 ml collagen]; BEI = 0.517 [0.2 ml collagen]), although the differences were not statistically significant. Hepatocytes cultured in MCM produced consistently the lowest BEI of TC (BEI = 0.396). Differing DEX concentrations (0.1 μM versus 1.0 μM) with or without 5% FBS did not appear to have a significant effect on the BEI of TC.

The West-Indian manatee, Trichechus manatus latirostris, is a herbivorous marine mammal found in the coastal waters of Florida. Because of their endangered status, animal experimentation is not allowed. Therefore, a cell line was developed and characterized from tissue collected during necropsies of the manatees. A primary cell culture was established by isolating single cells from kidney tissue using both enzymatic and mechanical techniques. Primary manatee kidney (MK) cells were subcultured for characterization. These cells were morphologically similar to the cell lines of epithelial origin. An immunocytochemistry assay was used to localize the cytokeratin filaments common to cells of epithelial origin. At second passage, epithelial-like cells had an average population-doubling time of 48 h, had an optimum seeding density of 5 × 10³ cells/cm², and readily attached to plastic culture plates with a high level of seeding efficiency. Although the epithelial-like cells had a rapid growth rate during the first three passages, the cloning potential was low. These cells did not form colonies in agar medium, were serum dependent, had a limited life span of approximately nine passages, and possessed cell-contact inhibition. These data suggest that the cells were finite (noncontinuous growth), did not possess transformed properties, and were of epithelial origin. These cells are now referred to as MK epithelial cells.