Human anterior pituitary adenoma cell attachment in vitro

Abstract

Dear Editor:The purpose of this letter is to inform fellow scientists of a method for the attachment of enzymatically dispersed human anterior pituitary adenoma cells, which often adhere poorly to the surface of cell culture dishes preventing dynamic in vitro studies: this is especially recognized for prolactin secreting and nonfunctioning pituitary adenomas (3,10-12). Attempts to enhance pituitary adenoma attachment have included the use of an extracellular matrix derived from bovine corneal endothelium, which has been reported to be successful for prolactinomas (3) and Cushing's adenomas (15). It has also been reported that rat pituitary endocrine cells express laminin and collagen IV, and that these cells grow as small clusters on fibroblast monolayers, which in turn are composed of collagen IV, heparin sulphate, and glycoprotein (13). Others have shown that the extracellular matrix components present among epithelial ceils forming Rathke's pouch consist of laminin, fibronectin, and collagen IV (8). Therefore, we set out to determine the optimal conditions for the attachment of different human anterior pituitary adenoma subtypes in dispersed culture using a panel of attachment factors: an extracellular matrix derived from bovine corneal endothelial cells, collagen type I and IV, fibronectin, laminin, human extracellular matrix (HEM), poly-l-lysine, plastic, and glass.Sixteen human pituitary adenomas--three macroprolactinomas, eight acromegalic adenomas, four nonfunctional tumors, and one corticotrophinoma removed by transsphenoidal surgery--were dispersed to single cells using a combination of a 200 mOsm hypoosmolar medium, clostripain inhibited crude collagenase, and dispase type 2 (1). Adenoma cells were plated at a density of 10 ~ cells per well in plastic 24-well plates with and without attachment factors in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% fetal calf serum (FCS) and gentamicin (50 rag/l). Cultures were maintained at 37 ° C in a humidity controlled atmosphere of 95% air and 5% CO2. After the cells had been allowed to adhere for 4 d, the total attachment was assessed using a calibrated graticule and viability was determined using fluorescein and propidium iodide (2), following which the wells were gently washed three times in IMDM to remove nonattached cells, and attachment and viability were reassessed. Four replicates were used for each group.Bovine corneal endothelial matrix (BCEM) were prepared from cells 7 d postconfluence: 1-6 d postconfluence gave a BCEM that was too thin, contained multiple holes, and peeled off from the culture surface; 9 d postconfluence the membrane was thicker, but peeled off after preparation. Three methods were compared for BCEM preparation: firstly, BCE cells were washed with phosphate-buffered saline (PBS) and 5 ml of 0.5% Triton X100 (Sigma Chemical Co., St. Louis, MO) (6); secondly, 0.1% Triton X100 ]adapted from (6)]; or thirdly, 20 mmol freshly prepared ammonium hydroxide (5) was added and incubated for 15-20 min and monitored by phase con...

Growth hormone receptor expression and function in pituitary adenomas

“…After 3 h in this medium containing collagenase, the tissue samples were dispersed into a single‐cell suspension and seeded into 96‐well plates (NUNC, Denmark) and 8‐well Chamber Slides (NUNC, Denmark), precoated with fibronectin (5 µg/cm 2 ). Media were supplemented with 10% FBS, l ‐glutamine (2 m m ), pentrexyl (0·1 g/l), gentamicin sulphate (50 mg/l) and fungilin (2 mg/l) (Doyle et al ., 1990; Atkin et al ., 1993, 1997b). Cultures were maintained at 37 °C in a humidity controlled atmosphere of 95% air and 5% CO 2 .…”

A human ACTH-secreting corticotroph tumoroid model

“…However, these human corticotroph tumor cells typically loose ACTH hormone production after 1-2 weeks [11] , [12] , [13] , [14] , [15] . Purported reasons for their loss of endocrine secretory function have included altered cell adhesion [ 11 , 16 ], lack of intrinsic hypothalamic factors [17] , cellular trans- or de-differentiation [18] and/or deactivation of prohormone convertases [19] . However, no clear changes in specific molecular candidates or pathways, that might provide insight into the mechanisms for loss of corticotroph tumor ACTH secretion, have been demonstrated.…”