Raymond H. Kahn

Sequential studies of healing in endothelial seeded vascular prostheses histologic and ultrastructure characteristics of graft incorporation

Abstract Chronological events leading to incorporation of endothelial cell seeded prosthetic vascular grafts were documented in this investigation. Forty-one adult dogs underwent thoracoabdominal bypass using double-velour Dacron grafts. Experimental grafts were preclotted with blood containing enzymatically derived endothelium immediately after derivation, or after 14 days of cultivation. Control grafts were preclotted without addition of endothelial cells. Grafts were studied grossly as well as by light, scanning electron, transmission electron, and fluorescence microscopy, 1 to 28 days postimplantation. Control graft healing proceeded from pannus and perigraft ingrowth. Experimental grafts healed from seeded cells as well. Platelets covered all grafts by Days 1 and 2. Thrombus accumulations on control grafts, first evident on Day 4, became maximal by Day 14. Seeded grafts appeared relatively thrombus free with patches of endothelial cells noted by 4 days. These cells were initially separated by gaps, often containing leukocytes. Endothelium became densely packed with cellular migration and proliferation. Subendothelial tissues were composed of fibrin and smooth muscle. Control and experimental grafts were approximately 10 and 80% endothelialized, respectively, by Day 28. Smooth muscle dominated subintimal tissue in experimental grafts. These cells initially appeared fibroblastoid. Endothelial seeding enhances both pseudointimal development and rapid graft incorporation.

Isolation of adult canine venous endothelium for tissue culture

SummaryIn order to provide autologous adult endothelial cells for the production of cell-lined artificial vascular prostheses, we have developed a method for harvesting large numbers of cells with minimal contamination by other cellular types. In this technique, the vein to be stripped is isolated, removed, and everted over a stainless steel rod. After washing, the vein is incubated in trypsin-EDTA solution followed by collagenase and the endothelial cells flushed off with a stream of culture medium. With care and appropriate timing, the endothelium can be selectively removed leaving the underlying basal lamina intact.

Histochemical studies of rat ovarian follicular cells in vitro.

SummaryMembrana granulosa cells were aspirated from large follicles of proestrous rat ovaries and were cultivated as monolayers. For histochemical identification of dehydrogenases, the monolayers were incubated in various steroid substrates, nicotinamide adenine dinucleotide, and Nitro Blue Tetrazolium. The presence of Δp5-3β-, 3α-, 17β- and 20α-hydroxysteroid dehydrogenases was demonstrated by the 4th day in vitro and was evident for as long as 20 days. Since none of these hydroxysteroid dehydrogenases is demonstrable in the membrana granulosa of intact follicles, it is concluded that the steroidogenic capacity of the cells, repressed in the preovulatory follicle in vivo, can be expressed upon mechanical removal from the follicle just as steroid synthesis occurs in these cells after normal ovulation.

Prolactin Production by Rat Pituitary in vitro.

Summary Anterior pituitary explants from female immature, mature and postpartum lactating rats were cultured in vitro on “199” or Trowell T-8 medium at 35 ± 1°C for periods of 6-14 days. Medium was assayed for prolactin by the intradermal pigeon crop method. Pituitary explants from mature female rats, gassed continuously with 95% O2 − 5% CO2 released 5-10 times more prolactin into medium each day than explants exposed only to air atmosphere. The amount of prolactin per AP recovered each day from the culture medium, under continuous gassing, was 2-3 times greater than that present in a fresh AP prior to culture. This proves that prolactin was actively synthesized and released and not merely “leached” out from AP explants. Explants from immature rats elaborated about half as much prolactin per AP per day as explants from mature rats, and ex-plants from lactating rats about twice as much as explants from mature rats. Assays of some explants at the end of culture revealed a low prolactin content, indicating rapid synthesis and release. Histological examination of ex-plants at termination of culture showed that tissue exposed to continuous gassing was almost entirely viable, whereas tissue exposed to air atmosphere had only a thin outer rim of living cells. These results demonstrate that prolactin can be produced in large amounts when AP tissue is cultured in vitro.

Fiber formation in suspension cultures of L strain fibroblasts.

Summary The in vitro formation of fibrous membranes by the L and LLC-Ml strains of mouse fibroblasts in a protein-free medium has been demonstrated. When cells of either of these strains are suspended in medium 199 plus 0.5-1.0% peptone and incubated at 35°C on a rotary action shaker at 100 rpm, delicate membranes form at the liquid-air interface. Enzymatic digestion, staining reactions and electron micrographs suggest that the fibers are fibrous protein in nature and belong to the collagen class of protein. The possible relationship of this fibrous protein to the cell surface is discussed.

EFFECT OF TESTOSTERONE ON LONG-TERM ORGAN CULTURES OF CANINE PROSTATE

SummaryOrgan cultures of rodent and human prostate glands have shown marked differences in their morphological response to testosterone. In this study, explants from 19 canine prostate glands were cultivated for a minimum of 9 days in Trowell’s T-8 medium. Groups of explants were exposed to media containing from 0.05 to 100 µm testosterone. While the higher testosterone levels (50 and 100 µm) markedly decreased explant viability, explants cultivated at lower levels (0.05 to 5 µm) appeared similar to control explants in testosterone-free Trowell’s T-8 medium. Atmospheric mixtures containing either 95% or 50% oxygen were equally effective.Shortly after the cultures were initiated, large amounts of secretory product were liberated into the lumen. After 9 or more days in vitro, glandular epithelium appeared cuboidal and never revealed the acid phosphatase-rich secretory granules seen in the preculture control. However, the epithelium exhibited an increase in alkaline phosphatase and lipid content following cultivation.

PROPAGATION OF PSEUDO-INTIMAL LININGS OF VASCULAR PROSTHESES

SummaryIn an attempt to develop viable intimal linings for artificial vascular prostheses, an investigation was undertaken of methods to ensure cell deposition and adherence to microfabric-lined devices, and to stimulate cellular propagation on these surfaces.The WI-38 cell was used as a model. Technical procedures developed for seeding, adherence, and growth included: (a) degassing of the microfabrics before seeding to permit penetration of media, (b) centrifugation to seed cells forcibly into the microfabric, (c) containment of cells during the early stages of in vitro cultivation to prevent outgrowth of the newly seeded cells, and (d) perfusion of the cultures on a fixed schedule for removal of waste products and replacement with fresh media.Of the parylene C-coated polypropylene microfabrics tested, the nonvertically drafted, microwave discharge-treated fabrics were decidedly better suited for cell attachment of the WI-38 cell than multiple drafted or maximal drafted fabrics which provided less adequate surfaces for cell attachment. Although the adhesive used in cementing the microfabric to a backing is undoubtedly toxic, parylene C coating protects the cells. Nylon microfabrics tested to date proved deleterious to confluent cell growth. Gold fabrics were highly compatible with the cells.

Ovarian Histology After Treatment of Rats with Norethynodrel.

Summary Norethynodrel caused enlargement of the corpora lutea with cellular hypertrophy and depletion of sudanophilic lipid and cholesterol. These changes appeared to be mediated by the hypophysis through the accelerated secretion of prolactin. On the other hand, ovarian interstitial tissue underwent involution with depletion of sudanophilic lipid and cholesterol. These effects were probably mediated by suppressed secretion of LH by the hypophysis.

Vernon P. Perry, LCDR MSC USN (Ret) (1927–1990)

Vernon Perry died on October 16, 1990 after a lifetime commitment to his family, his country and science. As a young Hospital Corpsman, Vernon was assigned by the U.S. Navy to the Tissue Bank at the National Naval Medical Center, Bethesda, MD. In that chance event, Vernon embarked on an amazing career based on his unique ability to envision, plan, organize and manage. His Commanding Officer, Dr. George Hyatt, aware of the importance of tissue culture to the Navys research and development program, arranged for Vernon to be assigned to the laboratory of Dr. Wilton Earle at the National Cancer Institute where our lifelong friendship with Vernon began. Apropos to his work for the Navy Tissue Bank program on transplantation, Vernon was a coauthor of a series of publications with Earle and his staff relating to the culture of humap skin. Vernons continued responsibilities at the Tissue Bank encompassed several developmental programs culminating in the successful operation of a skin and bone bank. It was at this time that Vernon initiated correspondence with Charles Lindberg, resulting in Lindbergs agreement to visit Vernon at the Tissue Bank. Vernon and Lindberg collaborated (1966) in developing an improved model of the perfusion pump which Lindberg had earlier designed and built for the Nobel laureate surgeon, Alexis Carrel. Vernons experiences in cell, tissue and organ culture inspired him to bring together workers in related life sciences. Itis collaborative studies with Fr. Basil Luyet and other investigations in low temperature biology led to the formation of the Society of Cryobiology and to the organization and establishment of the journal Cryobiology. Similarly, his work at the Navy Tissue Bank gave early recognition to a need for tissues and organs for transplantation. Again Vernon Perry was one of the prime movers in the formation of the American Association of Tissue Banks, and he served that organization for many years. After retiring from the Navy, Vernon Perry became the Director of the Biomedical Research Institute, Rockville, MD, a division of Ft. Basil Luyets American Foundation for Biological Research. Vernons greatest interest was the Tissue Culture Association. In his own quiet way, Vernon had four ambitions for the TCA which he helped the association to attain, namely its own journal, continuity for the Decennial Review Conference, an annual bibliography and a manual of tissue culture methods and procedures. When the TCA decided to publish its own journal, Vernons earlier experiences and personal efforts were central to the establishment and publication of In Vitro. He worked especially hard for the W. Alton Jones Cell Science Center and during the early years (1973-74) served as its Acting Director, commuting between Lake Placid, NY and RockviUe, MD. While the bibliography became outmoded with the advent of computers and has been discontinued, the Cell Science Center, In Vitro and the Decennial Review Conferences are testimonies to his commitment and personal efforts. Vernons interests in and concern for the TCA covered a period of more than forty years. For those of us who knew him well, Vernon was one of those rare individuals who worked quietly behind the scenes and contributed significantly to every aspect of the Associations activities. In the dignified way that he conducted his life, Vernon Perry has left an indelible influence on many of his coworkers in the fields of cell, tissue, organ culture, cryobiology and transplantation. Vernon is survived by his wife Esther, their five children, Richard, Paul, Fred, Michael and Mary Ellen Maher, twelve grandchildren, and two brothers.