Jeffery R. Cook

Microporosity of the substratum regulates differentiation of MDCK cells in vitro

SummaryWe have analyzed the ability of the physical substratum to modulate both the ultrastructural and protein synthetic characteristics of the Madin-Darby canine kidney (MDCK) renal cell line. When MDCK cells were seeded on Millipore Millicell CM microporous membrane cell culture inserts they demonstrated a more columnar organization with an increase in cell density sixfold greater than the same cells seeded on conventional plastic substrata. After 1 wk postseeding on the microporous membrane a partial basal lamina was noted, with a contiguous basement membrane being apparent after 2 wk. One-dimensional sodium dodecyl sulfate gel electrophoresis was used to analyze detergent-solubilized proteins from MDCK cells maintained on plastic substrata vs. microporous membranes. When proteins were pulse-labeled with [35S]methionine, a 55 kDa protein was evident in the cytosolic extract of cells grown on collagen, laminin, and nontreated plastic substrata; but this labeled protein was not evident in similar extracts from cells grown on collagen and laminin-coated microporous membranes. To test if the polarized, basement-membrane secreting phenotype of the MDCK cells could be generated on a microporous membrane without pretreatment with any extracellular matrix (ECM) components, cells were seeded on the Millipore Millicell HA (cellulosic) microporous membrane. This type of substrata does not need a coating of ECM components for cell attachment. A partial basement membrane was formed below cells where the basal surface of the cell was planar, but not in areas where the cell formed large cytoplasmic extensions into the filter. This led us to the conclusion that the microporous nature of the substrata can dictate both ultrastructural and protein synthetic activities of MDCK cells. Furthermore, we suggest that both the planar nature of the basal surface and the microporosity of the substrate are corequisites for the deposition of the basement membrane.

A Human Epidermal Model can be Assayed Employing a Multiple Fluorescent Endpoint Assay and the Cytofluor 2300

AbstractA human epidermal model (HEM) was developed that could be rapidly and automatically assayed in the CytoFluor 2300 (Millipore Corporation, Bedford, MA) spectrofluorometer using multiple site-and activity-specific fluorescent probes. The HEM was cultured on the optically translucent Millipore Millicell CM microporous membrane. Application of a variety of fluorescent dyes to this membrane without the HEM revealed negligible nonspecific dye association. The HEM was differentiated on a cross-linked collagen matrix and the latter was also found to retain less dye than the HEM. Feasibility experiments using the site/activity-specific dyes calcein-AM (plasma membrane integrity indicator), sodium fluorescein (epidermal permeability indicator), 5-chloromethylfluorescein diacetateacetoxymethyl ester (CMFDA-AM; intracellular glutathione level indicator), rhodamine 123 (mitochondrial activity indicator), neutral red (lysosomal integrity indicator), Fluo3-AM (intracellular calcium indicator), and a similar huma...

Cold-Storage of Synthetic Human Epidermis in HypoThermosol

There is a growing need for engineered tissues in a wide variety of medical applications and as alternatives to animal tissues for in vitro toxicology testing. While techniques for the preparation of a variety of synthetic tissue constructs have been devised, little attention has been focused upon the optimum conditions necessary for storage and shipping of these tissue devices. This study investigates the effects of hypothermic storage on synthetic human epidermis (EpiDerm, MatTek Corp., Ashland, MA) and specifically examines the quality of storage in keratinocyte growth medium (KGM), a standard skin culture medium, compared with storage in HypoThermosol, a new hypothermic preservation solution. EpiDerm samples were immersed in HypoThermosol for 1 to 13 days at 4 degrees C and were assayed using the noninvasive, viability indicator dye, Alamar Blue (AB). Samples immersed for 1 to 9 days in HypoThermosol retained their viability subsequent to warming to 37 degrees C and for at least 7 days thereafter in culture. During this time samples previously stored in HypoThermosol continued to generate a stratum corneum and their ultrastructural characteristics were similar to EpiDerm that were not exposed to hypothermic solutions. This profile, however, was not apparent in EpiDerm maintained for 1 to 13 days in KGM and subsequently warmed. These samples appeared viable immediately upon warming in most cases, but viability was not retained thereafter. EpiDerm maintained in KGM and allowed to recover at 37 degrees C appeared necrotic and failed to continue to differentiate. The conclusions of this study are the following: (1) HypoThermosol protects the viability of EpiDerm during cold-storage, (2) HypoThermosol preserves EpiDerms ability to differentiate subsequent to warming, and (3) the inferior preservation of samples stored in KGM was most apparent 24 h after warming.

Matrix and laminin synthesis in MDCK cells in vitro.

Dear Editor: The Madin-Darby canine kidney cell line (MDCK) has been used for over 30 yr to study cell attachment (8), viral budding (11), transcytosis (4), transepithelial electrical resislance (1), polarized protein sorting and release (5,6), basal lamina deposition (2,3,7), and cell migration (12). These apphcations are possible, in pall, because MDCK cells grown to conflueney form a contiguous, polarized monolayer linked by tight junctions (2,7). Due to the presence of these tight junctions, MDCK ceils are also being used in in vitro toxicology to assess ocular irritancy. For instance, several investigators have clearly shown that it is possible to rank irritants based on the ability of sodium fluorescein to leak through an irritant-exposed, MDCK monolayer (9,10). A mandatory component of this assay as well as most of the other aforementioned investigations is a microporous membrane cell culture insert. Microporous membranes have been used extensively as semipermeable support devices to grow MDCK cells and other epithelial cell lines. Cook et at. (2) have shown that a microporous support facilitates the deposition of a basal lamina underlying MDCK cells. This extracellular matrix structure does not, however, appear if MDCK cells are grown on plastic cell culture dishes. With the widespread use of microporous membranes in MDCK cell culture it is unclear how, if at all, these cells respond biochemically to subtle differences to either the type of microporous membrane or the type of extracellular matrix apphed to the membrane to enhance cell attachment. The data presented in this letter indicate that MDCK cells can respond to differences in three of the five substrates tested, as reflected by changes in the synthesis of the basal lamina component, laminin. The five subs/rates consisted of combinations of microporous membranes, dry collagen films, and collagen gels. Dry collagen films were prepared as follows. Aqueous rat tail type I collagen (Collaborative Research, Bedford, MA) was diluted 50:50, vol/vol, with 100% ethanol. Thirty-millimeter Millipore Millicell CM microporous membranes (Millipore Corp., Boston, MA) and 35-mm Falcon cell culture dishes (plastic substrate) were coaled with this mixture and designated DCF-CM and DCF-P, respectively. The excess collagen was removed and the cell culture dishes and microporous membranes were allowed to dry overnight. Afterward, the dried collagen was cross-linked with 2.5% glutaraldehyde (Ladd Scientitle, Burlington, VT) in phosphate buffered saline (PBS) for 1 h. Next, subs/rates were rinsed 3 times in PBS and either used immediately or stored in cold PBS until needed. Collagen gels were prepared as follows. Rat tail type I collagen (Collaborative Research) was diluted 2:1, vol/vol, with 70% ethanol. Five hundred microhters of this mixture were added to the inside of a 30-mm Millipore Millicell CM microporous membrane and 587 ~1 were added to the surface of a 35-mm Falcon cell culture dish. These substrates were referred to as cross-hnked type I collagen gel-covered Millipore Millieell CM microporous mem-

A HUMAN EPIDERMAL MODEL GROWN ON AN ACELLULAR GEL

AbstractAn in vitro epidermis was constructed from normal human epidermal keratinocytes (NHEK) grown on a cross-linked, acellular, type I collagen gel. The latter was overlaid on a Millipore Millicell CM microporous membrane. This human epidermal model (HEM) was differentiated in the presence of elevated levels of calcium and raised to the air-liquid interface. The HEM differentiated into 20+ cell layers and demonstrated ultrastructural characteristics of human epidermis in vivo, including an electrondense stratum corneum, keratin filaments, desmosomes, lamellar-like bodies, and, most notably, a structure resembling the basal lamina at the interface of the epidermis and the gel. This basal lamina appeared in preparations grown at either the air-liquid interface or under more conventional, submerged conditions. A planar basal surface was a corequisite for the appearance of the basal lamina in both cases. Because this HEM is one of the few in vitro epidermal models reported to construct a structure resembli...

A Double-Label Technique that Monitors Sulfur Mustard Damage to Nuclei and Mitochondria of Normal Human Epidermal Keratinocytes In Vitro

Sulfur mustard and 2-chloro ethyl ethyl sulfide (CEES, a sulfur mustard analog) is known to have immediate (minutes), long-term (hours to days), and toxic effects on human skin. Research was directed toward developing a single in vitro assay that might reflect both these short-term and long-term effects of this vesicating agent on normal human epidermal keratinocytes (NHEK) in vitro. Such an assay system would be useful in identifying and developing sulfur mustard therapeutic agents. NHEK were exposed to the monofunctional sulfur mustard analog 2-chloro ethyl ethyl sulfide for a variety of times. The effects of CEES on NHEK nuclei were assessed using the membrane-permeable SYTO nuclear stains, whereas the effects of CEES on NHEK metabolism were determined by using the nontoxic mitochondria dye Alamar blue. CEES enhanced SYTO binding in a concentration-dependent manner to the nucleus immediately subsequent to a 2-hr exposure, whereas CEES had relatively little effect on metabolic activity at this time. Fifteen to 36 hr subsequent to CEES exposure, however, Alamar blue revealed a robust, sulfur mustard-dependent effect on mitochondrial activity. To determine if both these indicator dyes could be used simultaneously, NHEK were exposed to CEES and stained with the SYTO nuclear stain 2 hr subsequent to exposure. This procedure was followed by assay of the same cell cultures with Alamar blue at 36 hr subsequent to initial CEES exposure. The data indicate that this nuclear/mitochondrial double-label technique can be used to monitor the short- and long-term effects of sulfur mustard on the same culture of NHEK.

The matrix form of collagen and basal microporosity influence basal lamina deposition and laminin synthesis/secretion by stratified human keratinocytes in vitro

SummaryThe ability of the collagen matrix form to support the formation of a basal lamina by cultured normal human epidermal keratinocytes (NHEK) was determined using transmission electron microscopy. The collagen matrix forms tested in this study were a) a dry type I collagen film and b) a type I collagen gel. NHEK were grown for 14 days on the following five different substrates: plain plastic culture dishes without the addition of collagen (PP); plain plastic culture dishes overlaid with a dry, aldehyde-crosslinked type I collagen film (DCF-P); plain plastic culture dishes overlaid with an aldehyde-crosslinked type I collagen gel (GEL-P); Millipore Millicell CM microporous membranes overlaid with a dry, aldehyde-crosslinked type I collagen film (DCF-CM); and Millipore Millicell CM microporous membranes overlaid with an aldehyde-crosslinked type I collagen gel (GEL-CM). NHEK maintained for 2 wk on PP and DCF-P were unable to secrete a basal lamina. NHEK grown for 2 wk on the GEL-P and GEL-CM substrates, however, secreted a contiguous basal lamina at the GEL-NHEK interface. To determine if the appearance of this basal lamina correlated with laminin synthesis, laminin was immunoprecipitated from cellular extracts, as well as media from the apical and basal chambers. NHEK grown on the GEL-P substrate synthesized more laminin than did NHEK grown on the other four alternative substrates. In addition, NHEK grown on GEL-CM were able to direct more laminin to the basal compartment than NHEK grown on DCF-CM substrates. Taken together, the data indicate that the matrix form of collagen can influence basal lamina deposition, laminin synthesis, and laminin trafficking in NHEK.

Disruption of microfilaments alters laminin synthesis but not laminin trafficking in NHEK in vitro.

SummaryLaminin synthesis and deposition are concomitant with the development of a basal lamina between the human epidermis and the underlying dermis. One of the challenges in tissue engineering of human epidermal models is to develop substrates and conditions that encourage the development of a basement membrane. The purpose of this study was to determine if actin filaments and/or microtubules are involved in the synthesis/secretion of laminin by normal human epidermal keratinocytes (NHEK)in vitro. NHEK synthesize and secrete laminin subunits B1, B2, and M but little, if any, of laminin subunit A. Data indicate that disruption of microfilaments by the destabilizing agent, cytochalasin D, had no apparent effect on the relative synthesis rates of most cytosolic proteins as, revealed by one-dimensional sodium dodecyl sulfate (SDS) gel electrophoresis. This drug, however, increased laminin B2 synthesis several fold over untreated controls. This enhanced synthetic rate was independent of the type of collagen, matrix on which the NHEK were grown. Similar increases in synthesis of the M and B1 laminin chains were not observed. To determine if this increase in synthesis lead to increases in laminin B2 secretion, laminin B2 was immunoprecipitated from both the apical and basal domains of NHEK cells grown on microporous membranes. While more laminin B1, B2, and M were secreted basally than apically, an observation consistent with laminin’s role in basal lamina formation, cytochalasin D had no apparent effect on either basal or apical laminin B2 secretion. Experiments with the microtubule destabilizer, nocodazole, showed no similar effects on laminin synthesis and/or secretion. We conclude that (a) disruption of the actin network in NHEK selectively increases the synthesis of laminin B2, (b) the secretion of laminin B2 from NHEK cells is not governed by either the microfilamentous cytoskeleton or the amount of laminin synthesized by NHEK, and (c) disruption of the microtubular network does not alter laminin synthesis or secretion.