The study of growth and differentiation of mammary epithelium has been hampered by the difficulty of maintaining these functions in vitro. We describe a system for the primary culture of rat mammary epithelium on an acellular matrix derived from whole rat mammary glands that maintains growth and differentiation for months. Cultures plated on this complex substratum produce 50 times the ei-lactalbumin of those on tissue culture dishes and 5 times the a-lactalbumin of those on floating collagen gels as determined by radioimmunoassay. Unlike cultures grown on floating collagen gels, which rapidly lose the ability to secrete the milk sugar lactose, mammary cells on this matrix retain this ability for over 30 days in culture. The organ specificity ofthis mammary extracellular material is shown by the failure of extracellular matrix prepared from rat liver to support mammary differentiation. Within a given culture dish, cells on the surface of mammary extracellular matrix are more differentiated than those on the adjacent plastic. This is demonstrated by their increased a-lactalbumin content as shown by indirect immunofluorescence, and by their increased ability to bind fluorescein-conjugated peanut lectin. Cells on the surface ofthe matrix continue to synthesize DNA as determined by [3H]thymidine incorporation and autoradiography. Even when mammary epithelial cells are plated at low density, cell division continues until the matrix is covered with a confluent layer. We propose that the limited growth, differentiation, and survival of mammary cells in previously described in vitro systems may have been due to substrata that were inadequate to support these functions.The study of mammary growth and differentiation has been hampered by the lack of a suitable system that is capable of maintaining these functions in vitro. When normal mammary epithelial cells from rodents or humans are cultured on tissue culture plastic surfaces they undergo only a few rounds of cell division and rapidly lose differentiated function (1-3). Sometimes, continuous cell lines that are easy to manipulate in vitro can be established from these cultures (4, 5). However, because these cells are highly selected to proliferate under artificial conditions, their control mechanisms may have little relevance to those ofmammary cells in vivo. Organ culture has the advantage of maintaining more normal tissue orientations. However, these systems have limited viability and the presence of stromal cells makes quantitation of epithelial growth difficult (6, 7).It has been appreciated for some time that cell behavior in vitro may be influenced by placing cells on matrices of stromal collagen (8,9). More recently, Emerman and Pitelka described a system for the culture ofmouse mammary cells on floating gels of stromal collagen. Mammary epithelial cells isolated from midpregnant mice produced considerably more ofthe milk protein casein when plated on these floating collagen gels than when plated on attached collagen gels or tissue culture plastic dishe...

After storage in the liquid state at 4 C for up to three weeks, washing with sodium chloride solutions, and storage in a sodium chloride-glucose-phosphate solution for 24 hours at 4 C, dog red blood cells had excellent post-transfusion survival. After freeze-preservation with 40% W/V glycerol at -80 C or with 20% W/V glycerol at -150 C, thawing, washing with sodium chloride solutions, and storage in a sodium chloride-glucose-phosphate solution for 24 hours at 4 C, dog red blood cells had satisfactory recovery values in vitro, acceptable 24-hour post-transfusion survival and long-term survival values, and normal oxygen transport function. Controlled addition and removal of the cryoprotectant, glycerol, helped reduce the amount of osmotic damage to the red blood cells and enhanced freeze-preservation. Osmotic damage can also be prevented by warming the dog blood to a temperature of 22 +/- 2 C prior to centrifugation to concentrate the red blood cells and remove the plasma. This step enhances removal of the cold agglutinins. Another processing step used by the authors was to add a sodium chloride solution to the dog red blood cells before adding the glycerol solution in order to eliminate rouleaux formation.

Baboons were bled one-third of their blood volume and then transfused with an equivalent volume of compatible donor red blood cells with 160 per cent of normal 2,3-diphosphoglycerate (2,3-DPG) levels and improved capacity to release oxygen to tissue. The mixture of baboon donor-recipient red blood cells in the circulation had a 2,3-DPG level of 130 per cent of normal. After transfusion, the baboon's inspired oxygen was first lowered from 21 to 10 per cent to produce severe arterial hypoxemia with a PO2 tension of less than 40 mm Hg for two hours and then restored to 21 per cent. Lactic acidemia occurred when the alveolar oxygen tension was reduced so as to produce an arterial oxygen tension of less than 40 mm Hg, even though oxygen consumption was maintained. The data suggest that when red blood cells with normal or improved oxygen delivering capacity are transfused to patients, the alveolar oxygen tension should be sufficient to maintain an arterial oxygen tension of greater than 40 mm Hg.

The mechanism of erythropoietin (Ep) production after acute haemorrhage has been thought to be due to a reduction in blood volume and tissue perfusion leading to tissue hypoxia. In the present study we have evaluated the effect of acute haemorrhage in the rat on the acid-base status, the red cell affinity for oxygen in vivo, and Ep production. Within a few hours after acute blood loss there was a respiratory alkalosis with an increase in blood pH, a decrease in pCO2 and an increase in the red cell affinity of Hb for oxygen in vivo that was temporally related to an increase in Ep production. Within 24 h after the acute haemorrhage, the blood pH AND PCO2, red cell affinity for oxygen in vivo, and Ep level returned towards normal. The decrease in in vivo red cell affinity for oxygen was associated with an increase in red cell 2,3-DPG levels and a decrease in Ep production.