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The method may further comprise removing the culture medium from the cell culture, re-feeding the cell culture with a serum-free medium with glucose, and measuring proinsulin production C-peptide production, or insulin release. For example, a naturally-occurring cell present in its natural living host is not isolated, but the same cell, separated from some or all of the coexisting materials in the natural system, is isolated. Such cell or cell populations could be part of a cell culture or cell population, and still be isolated in that such culture or population is not part of its natural environment.
The expansion of primary human pancreatic cells in these conditions produces cultures with a mixed epithelial IP phenotype, suitable for in vitro studies of IP cells for a variety of purposes, and suitable for transplantation in vivo for cell therapy for the treatment of diseases such as diabetes. Furthermore, the cells may be further cultured to yield insulin-producing cells as demonstrated below. IB , and CK19 Fig.
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IC and the composition of the cell pellet of freshly isolated primary human pancreatic cells Fig. Note epithelial morphology. Note superior growth when Collagen I surface is combined with the media formulation described herein, yielding results superior to the combination of Matrigel and commercial media with serum. Amylase is red, CK19 is green. Images were analyzed to determine total cells Figure 10A, blue nuclei and total positive cells Figure 10B, blue nuclei surrounded by green staining for CK Blue portions are DAPI stained nuclei.
However, the invention is not intended to be limited to the specific terminology so selected. It is to be understood that each specific element includes all technical equivalents, which operate in a similar manner to accomplish a similar purpose. Each reference cited here is incorporated by reference as if each were individually incorporated by reference. Examples are collagen, vitronectin, fibronectin, laminin. IP cells: Intermediate progenitor cells derived from an epithelial cell, such as, e. Expansion and transdifferentiation of primary acinar cells into glandular epithelial cells Culture Phase I.
Primary human pancreatic acinar cells are collected as waste from standard COBE gradient preparation of islet cells for transplantation Lake et al. After density gradient centrifugation, the islets are present as a layer between 1.
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Cell number and viability is assessed by trypan blue exclusion and enumeration on a hemacytometer by light microscopic observation. A preparation of starting material was formalin fixed and paraffin-embedded as a cell pellet about 24 hours after initial harvest of the pancreatic cells. Paraffin sections were prepared, placed on slides, and subjected to immunocytochemical analysis with antibodies to insulin Biogenex, San Ramon, CA , CK19 Biogenex , and Amylase Biogenex.
A minimum of 3 sections per sample were assessed with each marker. All antibody staining was carried out according to the manufacturer's suggestion with pre-diluted commercial antibodies.
EP1507848A4 - Expansion and transdifferentiation of human acinar cells - Google Patents
For CK19, a 3 minute treatment with pepsin enzyme Biogenex preceded the blocking step for the purpose of antigen retrieval. Briefly, the sections were rehydrated through graded ethanols, followed by a 15 minute incubation in phosphate buffered saline PBS without calcium and magnesium. Protein Blocker Biogenex was added for 30 minutes prior to addition of primary antibodies. After 3 5-minute washes, biotinylated secondary antibody Biogenex was added at a 1: This cell pellet Figure IC was comprised of 1. Replicate cultures were harvested at 3 day intervals via trypsinization and live cells as determined by trypan blue exclusion and enumerated on a hemocytometer.
The results shown in Figure 2 demonstrate that the serum-containing medium formulation described herein is superior to commercial media formulation for growth and maintenance of primary pancreatic cells. Figure 6D compares the results of expanding the cells in the various media; Figures 6A, 6B and 6C show high power images of the cell cultures expanded under the various media conditions. ECMs were placed into solution at the above concentrations and allowed to coat tissue culture-treated polystyrene surfaces according to manufacturer's suggestions of 1 hour at room temp.
Excess ECM solution was then removed and surfaces were rinsed twice in water. HamsF12 mixture 1: Cells were seeded onto tissue-culture polystyrene surface as a control. After 18 hours, unattached cells were washed away and remaining attached cells were re-fed with PCM and allowed to grow for 7 days prior to evaluation.
Cells were counterstained with DAPI fluorescent blue nuclear stain to visualize individual cell nuclei for cell counting. The metabolic activity of cells subjected to the various conditions was determined by an MTS assay.
The results of this analysis are shown in Figure 7. Unattached cells were washed off after 18 hours, and cultures were re-fed and allowed to grow for 8 days. The results are shown in Figure 4A-B. Tissue culture medium PCM was prepared as described above. Cells were seeded onto collagen-coated flasks cm 2 at 1.
Cultures were monitored over time by metabolic assay MTT and by trypsinization and cell counting, to establish cell number see example 3. Cell phenotype at the end of the culture period was assessed as follows: After formalin was removed and monolayers were rinsed, cultures were subjected to immunocytochemistry as described in previous section for CK19, amylase, insulin, and vimentin a marker of fibroblasts.
After formalin was removed and monolayers were rinsed, cultures were subjected to immunocytochemistry as described in previous section for CK19 and vimentin a marker of fibroblasts. Cells were also stained with amylase antibodies, but did not produce positive results due to release of digestive enzymes, such as amylase, by the cells over time in culture.
Acquisition of ductal markers by acinar cells was verified by demonstrating concomitant expression of CK19 and amylase in cell subpopulations during days of culture see example 5. For these experiments, CK19 primary antibodies were reacted with formalin- fixed cell cultures, followed by visualization with Alexaconjugated Goat anti-mouse IgG Molecular Probes.
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Then, cells were subjected to a blocking step Protein Blocker, BioGenex , followed by application of the second primary antibody anti-amylase. Images were collected as described above. Light microcopic observations were made daily. At the hour timepoint, dishes were sacrificed and stained with trypan blue to assess viability.
The results are shown in Table 3. TABLE 3. The results are shown in Figures 5A and 5B. Primary pancreatic acinar cells were cultured for several days in a 1: After 2 days of culture 4 days ex vivo , expression of amylase by the acinar cells is still strong Figure 8A, upper left panel, red staining as determined by immunocytochemistry.
Expression of CK19 is also apparent Figure 8B, lower left panel, green staining. Daily evaluation of cultures Figure 9 demonstrated that onset of CK19 expression begins around Day 2 of culture and by Day 5 cultures have lost most immunodetectable amylase expression and CK19 expression is prevalent. After approximately 7 days of culture using conditions described herein, the acinar cells have concrete ductular characteristics, now referred to as IP cells. IP cells were obtained by culturing primary acinar cells in a cell culture system comprising PCM and a Collagen I surface.
Monolayer cultures were rinsed 2x with PBS, then detached from the flasks with 0. Cells were pelleted by centrifugation at 1, RPM for 3 minutes in a swinging bucket centrifuge. Cell pellets were resuspended and washed 2x in PBS before a final centrifugation at 1, RPM for 3 minutes as described above. The labeled probes from each sample were hybridized with the plastic human 8 K gene arrays for about 16 hours, the arrays were washed and imaged according to the Atlas array protocols.
The Atlas image 2. The transcript signals were then extracted from these aligned arrays using the Atlas Image 2. In a few cases, e. A summary of the expression by these cell populations of a selected set of genes is presented in Table 4. Table 4 contains a list of genes expressed in IP cells and a comparison of expression patterns in primary acinar cells and primary ductal cells. Transformation of glandular epithelial cells into insulin producing cells - Generating insulin-producing cells by differentiation of IP cultures Culture Phase m. In the following examples, the base medium is composed of a 1: The concentrations of these media supplements are listed in Table 1.
On Day 5, half of the medium is removed and replaced with an equal volume of fresh medium containing differentiation promoting factors. The cellular phenotype of the cells comprising these cultures was assessed by immunocytochemistry as described above using monoclonal antibodies to vimentin, pro-insulin, C-peptide, MUC-1, and CK19 See Example 10, below. See Example 9, below.
Cells that had been cultured in differentiation medium for days were washed 3x in PBS, then re- fed with either 1 base medium described above with 5mM Glucose, or 2 base medium with 22mM glucose. ELISAs were conducted using the standard range assay procedure according to manufacturer's specifications. Plates were incubated on a shaker during the assay and results were read in a Tecan spectrophotometric plate reader.
Total ng of insulin or C-peptide per well were calculated for each media condition, for both 5mM glucose media and 22mM glucose media See Example Over a period of days, the cells readily formed three-dimensional pod-like structures, clearly observable by light microscopy Figure Some larger pods detached from the culture surface after about days in culture, and remained viable, as determined by trypan blue exclusion.
The pod-like stmctures were hypothesized to be aggregations of insulin-producing cells, and subjected to further analysis as described below. Figure 12B shows the same group of cells, many of which are positive for C-Peptide, which is produced when the proinsulin molecule synthesized within the cell is cleaved to yield mature insulin; the C-peptide stained cells are red, with a typical granular staining of the cytoplasm.
Figure 12C shows a higher power overlay image, demonstrating colocalization of CK19 and C-peptide in a small subset of cells. Co-stained cells appear yellow-orange on the overlay image. Cells cultured in base medium negative control , or in Combinations 1, 2 and 3 of the differentiation promoting media, were evaluated for their ability to release insulin and C-peptide into the culture medium.