Glycoprotein methods protocols - biotechnology 048-9-219-226.pdf

Glycoprotein methods protocols - biotechnology

18

Biosynthesis of Mucin Cell and Organ Culture Methods for Biosynthetic Study

Anthony P Corfield, Neil Myerscough,

Alexandra W C Einerhand, B Jan-Willem Van Klinken,

Jan Dekker, and Christos Paraskeva

1 Introduction

The study of the biosynthesis has been greatly assisted by the use of cultured cells and tissue explants in short-term culture Cells are available from a wide range of tissue sources, and this chapter focuses on the use of intestinal cells and tissue Human colonic cell lines have been widely used in biosynthetic studies and the relationship of some lines to stages in the adenoma-carcinoma sequence is of particular interest, allowing study of the expression of mucin during the development and progression of

disease (1–3) Recently the importance of proliferation, differentiation, and apoptosis

has attracted attention to the use of culture systems for the study of cell behavior in

normal and disease processes (4,5) In the same way, tissue obtained from patients at

surgery or as biopsies can be placed in short-term primary or organ culture to study

similar changes in disease (6,7).

Improvements in the study of glycoproteins, especially mucins, have been achieved through the use of defined human mucosal cells that can be grown in long-term culture

(1,2) Radioactive tracer methods allow relatively small numbers of cells and tissue

fragments (biopsies) to be analyzed, and cell culture also gives access to larger amounts

of the mucins produced by the individual cell lines (8,9).

Each of the model systems described for the study of the synthesis and secretion of mucin has distinct advantages and drawbacks Ideally we would wish for a clonal cell line, which has all the typical characteristics of the mucin-producing cells in vivo Because no such cell line exists for any of the mucin producing cells, we must settle for either tissue explants, in which the mucin-producing cells have retained their ral tissue context, or isolated clonal cells from carcinomas The first system is natu-rally short-lived, which affects reproducibility, whereas the latter system is far more reproducible; however, these cells will have irreversible genetic changes that

distin-From: Methods in Molecular Biology, Vol 125: Glycoprotein Methods and Protocols: The Mucins

Edited by: A Corfield © Humana Press Inc., Totowa, NJ

guish them from the cells in vivo On the other hand, the cell lines may consist of only one cell type, which can be an advantage.

Study of the colon has the advantage that the sequence of changes during carcino-genesis in the colonic epithelial cells has been documented particularly well This implies that cells can be isolated from each stage of malignant growth: starting from healthy tissue, from which normal epithelial cells can be brought into primary culture and display only minimal growth in vitro, via the adenoma stage that maintains inter-mediary characteristics to the full blown carcinoma cells, which usually grow quite

well (10) This sequence of changes in the epithelium allows precise studies of how

the changes in cells influence the mucins that are produced Several of the end prod-ucts of malignant transformation of colonic epithelium have yielded valuable cell lines, such as the goblet cell-like LS174T and the enterocyte-like Caco-2 cell lines, which

are widely used to study the synthesis and regulation of mucin in detail (11,12) The

PC/AA cell lines, originally derived from a single, large, colonic tubular adenoma, have been used at early premalignant, intermediate premalignant, adenocarcinoma,

and mucinous carcinoma stages (1,2,8,9,13) Furthermore, the HRA-19 colorectal cell line (3) is valuable because it can be cloned to give all three differentiation pathways

to columnar, goblet, and endocrine cells.

The methods described here cover the use of cellular systems for the study of the biosynthesis of mucin which includes cultured cells (primary cultures and cell lines) and organ culture The data refer to human colorectal cells and tissue, but similar systems and principles apply to a wide range of other tissues where mucins are produced.

2 Materials

1 Human gastrointestinal (GI) biopsies or surgical tissue from stomach (corpus and an-trum), duodenum, jejunum, ileum, colon (ascending, transverse, descending, sigmoid, and rectum), or gallbladder Tissue obtained at surgery is dissected to isolate the mucosal layer and cut into small sections of approx 2–4 mm3 Endoscopically obtained biopsies are used without further manipulations

2 GI tissue explants of rat or mouse from stomach (corpus and antrum), duodenum, jejunum, ileum, colon (proximal, mid, and distal), or gallbladder (only in mouse) Full thickness explants, including the muscle layers are used cut to give segments of 2–4 mm2of mu-cosal surface

3 Cell lines:

a Swiss 3T3 cells obtained from the American Tissue Type Culture Collection (ATCC, Manassas, VA, cat no CCL92)

b LS174T, colonic adenocarcinoma cell line (the parental line obtained from ATCC, cat no CL 188)

c Caco-2, colonic adenocarcinoma cell line (from Prof G J Strous, Laboratory for Cell Biology, Utrecht, The Netherlands)

d A431, epidermoid carcinoma cell line (ATCC, cat no CRL 1555)

4 Fetal bovine serum (FBS) Batch testing is essential

5 Cell culture media

a Standard growth medium: Dulbecco’s modified Eagles medium (DMEM) containing

2 mM glutamine, 1 µg/mL of hydrocortisone sodium succinate, 0.2 U/mL of insulin,

100 IU/mL of penicillin, 100 mg/mL of streptomycin, and 20% of FBS

b Washing medium: The same as the standard growth medium, but with 5% FBS, double the concentration of penicillin and streptomycin, and 50 µg/mL of gentamicin

c Digesting solution: The same as the washing medium but containing 5% FBS together with 1.5 mg/mL of collagenase (Worthington type 4, Lorm Diagnostics, Reading, UK) and 0.25 mg/mL of hyaluronidase (type 1; Sigma, Poole, UK)

d 3T3 Conditioned medium: DMEM is supplemented with 10% FBS, 2 mM glutamine,

100 IU/mL of penicillin, 100 µg/mL of streptomycin, and put onto 24-h postconfluent 3T3 cell layers for 24 h After conditioning, the medium is filtered through a 0.2-mm filter (Nalgene, Milton Keynes, UK) and further supplemented to give 20% FBS,

1µg/mL of hydrocortisone sodium succinate, and 0.2 U/mL of insulin

e Cell growth medium: Dulbecco’s modified Eagles medium (DMEM, Gibco/BRL, Parsley, Scotland) containing 100 IU/mL of penicillin, 100 µg/mL of streptomycin, 3.7 g/L of NaHCO3, and nonessential amino acids (sterile 100X stock solution, Gibco/ BRL), supplemented with 20% of FBS (LS174T cells) or 10% of FBS for Caco-2 and A431 cells

f Mouse thymocyte culture medium: RPMI medium (Gibco/BRL) supplemented with

10% FBS, 2 mM of glutamine, 100 IU/mL of penicillin, 100 µg/mL of streptomycin

and 2 mM of glutamine.

6 Collagen (human placental type 4; Sigma) at 1 mg/mL is prepared in 1 part glacial acetic acid to 1000 parts sterile tissue culture grade distilled water and, and stored at 4°C

7 Dispase solution: Dispase (grade 1; Boehringer, Lewes, UK) is prepared in DMEM con-taining 10% FBS, sterile filtered, and stored at –20°C

8 MF-1 mice (Olac, Bicester, UK)

9 Trypsin 0.1% by weight in 0.1% EDTA in PBS

10 Acridine orange (Sigma)

11 Organ culture medium:

a Grid cultures: DMEM, containing 10% FBS, 10 mM of sodium bicarbonate, 2 mM of

glutamine, 100 µg/mL of streptomycin, 50 IU/mL of penicillin, 50 µg/mL of

gentami-cin, and 20 mM of HEPES, pH7.2

b Submerged cultures: Eagle’s minimal Essential medium (EMEM, Gibco/BRL), supplemented with: non-essential amino acids (sterile 100X stock solution, Gibco/ BRL), 100 IU/mL of penicillin, 100 µg/mL of streptomycin, 2 mM of L-glutamine Incubate under 95% O2/5% CO2

12 Carbogen gas: 5% O2/5% CO2

13 Culture dishes (Costar, Cambridge MA)

14 Airtight capped, transparent tubes (3–5 mL) with round bottom (Sarstedt, Nümbrecht, Germany)

15 Water bath at 37°C

3 Methods

3.1 Preparation of Collagen Coated Culture Flasks and Swiss 3T3 Feeder Cells ( see Notes 1–3)

1 To prepare collagen-coated flasks, coat tissue culture flasks (T2525 cm2) with a thin layer

of collagen solution (Subheading 2., item 6; 0.2 mg/flask), and allow to dry at room

temperature in a laminar flow hood for 2–4 h (see Note 4).

2 Grow Swiss 3T3 cells (Subheading 2., item 1) on collagen on plastic tissue culture flasks

in DMEM containing 10% fetal calf serum until they are 24 h postconfluent

3 Lethally irradiate the cells with 60 Kgray (6 mrad) of radiation, or treat with 10 µg/mL of mitomycin C (Sigma) for 2 h

4 Wash the cells and produce a single-cell suspension using a Pasteur pipet (wide aperture

to avoid shearing of cells) The cells can either be used immediately as feeders or be stored at 4°C as a single-cell suspension for up to 4 d (see Note 5).

3.2 Primary Culture-Enzyme Digestion ( see Notes 3, 6, and 7)

1 Wash the tumor specimens (adenoma and carcinoma) four times in washing medium

(Subheading 2., item 5b) and cut with surgical blades to approx 1 mm3in a small volume

of the same medium

2 Wash the tissue four times in washing medium, and place in digestion solution

(Subheding 2., item 5c) Pellet cells by centrifugation at 300g for 5 min Roughly put 1 cm3

in 20–40 mL of solution

3 Rotate at 37°C overnight (12–16 h)

4 Mix the suspension by using a Pasteur pipet to improve the separation of the epithelial elements from the stroma resulting from enzymatic digestion

5 Filter the suspension through 50-mm mesh nylon gauze, or repeatedly allow to settle out under gravity and collect the pellets The large clumps of cells and epithelial tubules (organoids that contain the majority of the epithelial cells) are separated from the single cells (mostly from the blood and stroma) and cell debris

6 Wash the cell pellets three times, and place in culture on collagen-coated T25 flasks in the presence of Swiss mouse 3T3 feeders (approx 1 × 104cells/cm2) at 37°C in a 5% CO2

in air incubator (13) In some situations, 3T3-conditioned medium (Subheading 2., item

5d) can be used instead of adding mouse 3T3 cells directly to cultures (see Note 5).

3.3 Long-Term Culture of Adenoma Cell Lines ( see Note 8)

1 Prepare culture conditions for adenoma cell lines as previously described for primary cultures

2 Carry out passage of adenoma cells as clumps of cells using sufficient dispase just to cover the cells, and incubate for approx 30 min at 37°C Remove the cells as a sheet, and pipet with a Pasteur pipet to remove them from the flask and to break up the sheets into smaller clumps of cells (13)

3 Wash the clumps of cells and replate under standard culture conditions Reattachment of cells may take several days, and during medium changing, any floating clumps of cells must be centrifuged and replated with fresh medium

3.4 Long-Term Culture of Clonal Carcinoma Cell Lines, Including LS174T, Caco-2, and A431 Cell Lines ( see Notes 8–10)

1 Grow the carcinoma cell lines in tissue culture plastics without collagen coating and 3T3 feeders

in DMEM supplemented with 10% FBS and 1 mM of glutamine (Subheading 2., item 5e).

2 Carry out passage as single cells using 0.1% trypsin in 0.1% EDTA (see Notes 9 and 10).

3.5 Apoptotic and Differentiating Cells

3.5.1 Isolation and Identification of Apoptotic and Differentiating Cells ( see Note 11)

1 During routine culture of cell lines, remove floating cells with the medium and pellet by centrifugation Most cell lines give rise to floating cells, the majority of which undergo apoptosis

2 Stain the cells with acridine orange at 5 µg/mL in PBS, which stains the DNA and allows visualization of the condensed chromatin of apoptotic cells Stain cells for 10 min and then observe with a fluorescent microscope using narrow-band FITC excitation (excita-tion wavelength, 450–490 nm; and barrier filter, 520–560 nm) Count at least 300 cells

3 Extract DNA from 106cells and electrophorese on 2% (w/v) agarose gel containing 0.1 mg/mL of ethidium bromide at 40 V until the dye front has migrated 3 to 4 cm Run the DNA from an equivalent number of attached cells as a control, and use dexamethasone

treated mouse thymocytes as a positive control for DNA laddering (see Subheading 3.5.2.

and Note 11).

3.5.2 Preparation of Thymocyte Cell Cultures ( see Note 11)

1 Sacrifice MF-1 mice (Subheading 2., item 8) at 2–3 mo, dissect out the thymus and

release thymocytes by pressing through sterile gauze

2 Suspend the thymocytes in thymocyte culture medium (Subheading 2., item 5f) at a

density of 106/mL and treate with 10–7M of dexamethasone.

3 Collect samples after 18 h incubation with dexamethasone

3.6 Organ Culture of GI Tissue

3.6.1 Grid Cultures (6) ( see Note 2)

1 Place the biopsies or explants, singly or up to six per dish, on lens tissue placed over a

stainless steel grid in culture dishes with a central well containing 2 mL of medium

(Sub-heading 2., item 11a) The orientation of the tissue is with the luminal surface uppermost.

2 Place dishes in an incubation oven connected with a continuous supply of carbogen gas

(Subheading 2., item 12) at 37°C

3.6.2 Submerged Cultures (14,15) ( see Notes 2, 12–15)

1 Place the biopsy or explants, up to a maximum of six, in a small, airtight capped tube in

100µL of EMEM (Subheading 2., item 11b) (see Notes 16 and 17).

2 Blow carbogen gas into the tube and seal the carbogen gas atmosphere by replacing the cap

3 Place the tube in a 37°C water bath

4 Notes

1 The production and secretion of mucins by cultured colonic cells should be examined using cells at different stages of confluency, because the stages of growth may alter the differen-tiation properties of the cells and therefore the amount and type of mucin produced

2 When using organ and primary cultures, it is important to consider the heterogenous nature

of the cell types in the tissue (i.e., stromal elements and lymphoid cells in addition to the colonic epithelial cells) It may not be clear which cell type is producing the glycopro-teins Identification of specific cell-located glycoprotein expression can be further

exam-ined using histological methods with chemical, lectin, or antibody stains or in situ

hybridization to identify the cellular origin of the mucin of interest (Chapters 3 and 27)

3 The primary culture techniques described can be used for normal adult colon However, these are not as reproducible as those with the adenomas and carcinomas, and there are more problems from contaminating stromal elements There are at present no normal

adult colonic epithelial cell lines, only adenoma (1) and carcinoma cell lines such as PC/ JW/F1 (13), HT29 (10), LS174T (goblet cell like), and Caco-2 (enterocyte like) (11,12).

4 Collagen-coated flasks are necessary to obtain efficient attachment of primary cultures and some adenomas and carcinomas to the flasks, and to retain the optimum

differenti-ated characteristics of the cells (13) “Tissue culture-tredifferenti-ated” culture plates (Costar)

(Sub-heading 2., item 13) can be used successfully for established carcinoma cell lines

with-out collagen coating

5 The use of 3T3 cell feeders requires controls to determine which cell type is producing the glycoproteins of interest This can be achieved using 3T3-conditioned medium in which the production of mucin is being assessed, or removing the 3T3 feeders from the flask once the epithelium has grown In addition, the 3T3 cells should be examined for the production of mucin under the culture conditions used

6 Colorectal adenomas invariably need digestion with enzymes because of their organiza-tion into well-differentiated glandular structures With carcinomas, it is possible to adopt

a nonenzymatic approach with surgical blades to release small clumps of tumour cells

that can be cultured (16).

7 When using all colonic cell lines, it is important to check the true colonic epithelial nature using a battery of markers, including antikeratin antibodies, ultrastructural analysis

show-ing the presence of desmosomes, and other colonic differentiation markers (10) This

verification is especially important with primary cultures and newly derived lines, but may also be important when culture conditions induce changes in cell behavior

8 Although many tumor cell lines, especially colon carcinomas, can be grown in simple media without 3T3 feeders and collagen coating, the colonic cells retain better differentiated pheno-types when using the more complex culture conditions described for primary cultures

9 LS147T, Caco-2, and A431 cells are cultured in DMEM as described under Subheading

2., item 5e at 37°C and 5% CO2 Caco-2 and A431 cells are passaged (trypsinised) and split 1:4 and the medium is changed every 2 to 3 d LS174T cells are split 1:2 and the medium changed daily

10 The three cell lines LS174T, Caco-2 and A431, together produce all gastrointestinal

mucins known at present: LS174T produces MUC1, -2, -5AC, -5B, and -6 (12); Caco-2 produces MUC1 and -3 (12) and A431 produces MUC4 (Van Klinken, Einerhand, and

Dekker, unpublished results) They serve as excellent cell lines for the isolation of the corresponding mucin precursors as detailed in chapters 20 and 21 In addition, the PC/AA cell lines produce only MUC 1 and MUC2 at early passage, but at later passage and in

later premalignant and malignant stages, they show de novo expression of MUC5AC,

MUC5B, and MUC6 (Corfield, Myerscough, and Paraskeva, unpublished results)

11 Proliferating cells in the bottom of the colonic crypts migrate to the upper half of the crypts, where they differentiate These differentiated cells migrate to the top of the crypt, and there is evidence that they die by apoptosis and that apoptosis may be the terminal stage of differentiation The relationship among proliferation, differentiation and apoptosis may be studied using the culture system described here We have shown that cultured colonic normal adenoma and carcinoma cells spontaneously die by apoptosis in vitro and that the levels of apoptosis can be modulated by dietary short-chain fatty acids

(butyrate, acetate, and propionate) and bile acids (5) During routine culture of colonic epithelial cells, some cells detach from the flask and float in the medium (4) These cells

contain condensed chromatin, which can be detected with acridine orange staining In addition, characteristic DNA laddering resulting from internucleosomal fragmentation can be seen after analysis of total cellular DNA Dexamethasone treated mouse thy-mocytes are a convenient source of cells to use as a positive control for DNA laddering

12 A mucosal biopsy is taken by endoscopy from a human individual with an otherwise intact organ A tissue explant is obtained from surgically resected human or animal tis-sue For human tissue, the mucosa can be relatively easily dissected from the muscle

layer due to its size In animal tissues, smaller sections of the entire wall of the organ are used, because these tissues are more fragile and dissection is difficult The presence of the muscle layer does not appear to affect the synthesis of epithelial mucins

13 Submerging the tissue segments in the culture medium is particularly advantageous when the secretion of mucin is studied The grid technique results in the accumulation of a mucous gel layer on the top of the tissue during prolonged incubations and requires care-ful collection as a separate fraction Upon submersion, the mucins are secreted into the medium, although some of the mucin may remain adherent to the epithelial tissue The submerged culture system, under basic nonstimulated conditions, leads to the recovery of 20% of the total MUC2 in the medium during a 4-h culture of human colonic biopsies This percentage is very reproducible when compared for a large number of patients (Van Klinken, Einerhand, and Dekker, unpublished results)

14 The submerged tissue culture technique has the advantage that the mucins are more efficiently labeled during metabolic labeling experiments compared to the grid culture system In other words a higher incorporation of radioactive precursor is found in the mucins prepared using the submerged technique if the same concentration of precursor is used in both culture sys-tems Moreover, the labeling is more economical because similar labeling levels can be achieved with smaller (5%) amounts of precursor owing to the smaller volume (0.1 vs 2 mL) Because prolonged incubations will certainly deplete the medium of nutrients the submerged technique in small volumes (0.1 mL/explant) is only applied to the metabolic pulse labeling of tissue The grid culture system is thought to mimic the in vivo state more closely, with reten-tion of the secreted mucus at the tissue surface Although lower levels of incorporareten-tion into

mucins are found, these are quite adequate for comparative studies in disease (6,7).

15 Standard media usually contain sodium bicarbonate as primary buffer (2.2 g/L), and are used only with a 5% CO2 atmosphere Alternatively, the bicarbonate buffer can be replaced with the nontoxic buffer HEPES, which enables incubation under an air atmo-sphere and does not require CO2 GI tissue is extremely sensitive to hypoxic conditions; therefore, the use of an oxygen atmosphere (carbogen gas: 95% O2/5% CO2) is preferred

to the 95% air atmosphere

16 Temperature control by a water bath is preferred to an incubator because the heat exchange

is much quicker and the temperature is prone to only very slight fluctuations This is particularly relevant for pulse/chase experiments because these involve relatively short

incubation periods and require rapid temperature equilibration (see Chapters 20 and 21).

17 If the incubation is part of a pulse/chase experiment, the EMEM should be depleted of the

compound used as a precursor to label the mucin (see Chapter 19, Subheading 3.1., and

Chapters 20 and 21)

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