Human Brain in Tissue Culture I. ACQUISITION, INITIAL PROCESSING, AND ESTABLISHMENT OF BRAIN CELL CULTURES DONALD H. GILDEN, MARY DEVLIN, ZOFIA WROBLEWSKA, HARVEY FRIEDMAN, LUCY BALIAN RORKE, DANIELA SANTOLI A N D HILARY KOPROWSKI The M u l t i p l e Sclerosis R e s e a r c h C e n t e r of T h e Wistctr I n s t i t u t e a n d Depnrtm e n t of N e u r o l o g y of t h e U n i v e r s i t y of P e n n s y l v n n i a , 36th Street n t S p r u c e , P h i l a d e l p h i n , P e n n s y l v u n i u 19104 a n d D e p a r t m e n t of P a t h o l o g y , Philadelp h i u G e n e r a l H o s p i t a l , P h i l n d e l p h i n , P e n n s y l v a n i a 191 0 4 , U . S . A .
This paper details the i n vitro techniques used to establish cells ABSTRACT in culture froin the brains of 40 patients, most of whom had chronic neurologic disease. The clinical and pathologic features of these patients are given. The success in establishing cell lines was dependent upon the origin of tissue (biopsy vs. autopsy), the site of removal from the brain, and various environmental and technical manipulations in vitro.
The search for transmissible or viral agents in patients with multiple sclerosis and other chronic neurologic diseases necessitated the acquisition of considerable quantities of brain tissue for growth in vitro. Within the past three years, the Wistar Institute has accumulated brain specimens at biopsy or autopsy of 40 patients with multiple sclerosis, amyotrophic lateral sclerosis, Huntington’s chorea, dystonia musculorum deformans, Batten’s disease, tuberous sclerosis, intention tremor, alcoholism, glioma, acute viral encephalitis (including herpes simplex encephalitis), subacute sclerosing panencephalitis (SSPE), post-infectious encephalomyelitis, acute hemorrhagic leukoencephalitis, and brain tissue from patients with or without systemic disease. This paper details the acquisition and initial handling of these tissues and includes a description of the effect of biopsy on the clinical course of patients with chronic neurologic disease. Also described are the success and failure rates of establishing brain cell monolayers in culture, comparing explanting and trypsinizing techniques in both biopsy- and autopsyacquired tissue, the ability of tissue from various areas of the brain (gray or white matter, “normal” or diseased areas, plaques in multiple sclerosis brain, and gray matter from patients with primary gray matter disease) to grow in culture, the establishment of initial cultures from non-parenchymal J . COMP. NEUR.,1 6 1 : 295-306.
areas of brain (choroid plexus and optic nerve), observations on the effect of pH, and various fetal calf serum (FCS) concentrations on the formation of monolayers, the effect of changes in nutrient medium on initial cultures, and the effect of specimen storage on the ability to grow cells out of brain tissue. MATERIALS AND METHODS
Acquisition of brain tissue. All biopsy material was removed by a neurosurgeon in the operating room under sterile conditions and immediately placed in tissue culture medium. Autopsy was performed as soon a s possible after death. Tissue culture media. The medium consisted of Eagle’s minimal essential medium (MEM) with phenol red and a 2 X concentration of vitamins. In general, the medium was adjusted at pH 7.2 by addition of 5.6% sodium bicarbonate. When it was desired to keep the medium at pH 8.0 or pH 7.6, MEM buffered with organic buffers and sodium bicarbonate was used (Croce et al., ’72). FCS which had been inactivated for 30 minutes at 56°C (Flow Laboratories, Rockville, Maryland) was added to produce a final concentration of l o % , and L-glutamine (Flow Laboratories, Rockville, Maryland) was added to a final concentration of 0.5%. Penicillin was added to produce a final concentration of 100 unitslml and Streptomycin was added to produce a final concentration of 0.05 mg/ml. 295
296
G I L D E N ET A L
Autopsy procedure. Following removal of the brain from the skull under clean but not sterile conditions, and with the dura intact, the brain was placed with the base down on a clean towel. At this point the dura was carefully removed and the convexities were wiped with 70% alcohol. A long brain cutting knife was then dipped into 100% alcohol and flamed until dry. After 15 to 20 seconds had been allowed for the knife to cool, a coronal section was made through the frontal region of the brain. Appropriate sections were then picked out of the cortex (gray matter just beneath the surface) and separate pieces of subcortical white matter were sampled. This material was kept sterile and immediately placed in tissue culture media. Then a more posterior area of the convexities was wiped with 70% alcohol, the brain cutting knife again sterilized in 100% alcohol, and the entire procedure repeated. Multiple coronal sections through the brain and brain stem were made in this fashion.
Multiple scleroszs brain. Old plaque areas of multiple sclerosis brain were readily identifiable by their brownish color, sharp borders, and varying degrees of tissue retraction (Peters, ’68). Acute plaques were more difficult to visualize because of their lighter salmon color, irregular borders, and absence of retraction (Peters, ’68). Small 1-2 mm’ sections were removed from the center of the plaque, the junction of plaque and white matter (periplaque), and grossly “normal” white matter 1-2 cm from the plaque. White matter material was sampled from both fresh and old plaques (fig. 1) and also gray matter. Each specimen was processed separately. Explants. Although the sizes of the brain specimens placed in tissue culture medium varied, no more than 1 m m 3of tissue was usually removed. All explanting was done in a laminar flow hood (Biogard, Baker Co., Inc., Sanford, Maine). The brain specimen was cut into as small pieces as could be cut with sterile knives and the
Fig. 1 Grossly visible plaques in the cerebellar white matter of a patient with multiple sclerosis. Note chronic plaque (above) with dark rusty appearance and sharp borders in contrast to fresh plaque (below) with lighter salmon color and hazy borders. Plaque-white matter j u n c tion noted by short arrow and periplaque white matter noted by long arrows.
297
H U M A N BRAIN I N TISSUE C U L T U R E
pieces were washed once or twice in tissue culture medium, and seeded sparsely into a small glass petri dish and at least 2 T-25 plastic Falcon flasks in MEM with 50% FCS. No less than three explants were made from each specimen removed. The T-25 flasks were placed in a 37°C incubator with 4%-5% C 0 2 . Caps were kept loose all the time until a monolayer was achieved. Medium was not changed until one week after the initial explant was placed in the culture flask or petri dish. Further medium changes were done at approximately 7-10 day intervals thereafter. Trypsinization. Brain tissue to be trypsinized was rinsed twice in tissue culture medium (MEM plus 10% FCS) and placed in a 35 ml trypsinization flask (Bellco Glass Inc., Vineland, New Jersey). Trypsin was then diluted to a final concentration of 0.25% in phosphate buffered saline and FCS was added to make a final concentration of 3% (trypsin was purchased from Flow Laboratories, Rockville, Maryland, and was kept at a 2.5% concentration in modified Hank’s balanced salt solution without calcium and magnesium salts). This brain tissue was stirred for 15 minutes at room temperature. The supernatant was then removed and kept at 4 “C. An additional 10 ml of 0.25% trypsin was added to the brain tissue which remained and stirred again for 15 minutes at room temperature. The second supernatant was removed and pooled with the first. Both were kept at 4°C and the entire procedure was repeated a third time. All the suspended cellular material was spun at 800-1000 rpm for 5 minutes. The supernatant was removed and the pellet was resuspended in MEM plus 50% FCS. This entire material was then filtered through a nylon mesh and distributed to tissue culture flasks. All explanted and trypsinized brain tissue was kept in MEM with 50% FCS for no less than two weeks (at least one medium change). Thereafter, 50% FCS was replaced by 20% FCS until a monolayer was established. Lesser concentrations of FCS were not used in this laboratory until after the first cell transfer. All old medium was removed during each medium change, or half the volume of “spent” medium was removed and replaced with fresh medium. All explanting and trypsinizing procedures were performed in a laminar flow hood.
RESULTS
T h e effect of biopsy on the clinical course of patients In 19 out of 22 patients on whom biopsies were performed, there was no adverse effect on the clinical course of the patient. Most patients already had considerable neurologic deficit as a result of their disease (tremor, paralysis, dementia, and sometimes coma), and a n increase in neurologic signs was not observed after biopsy. One patient with demyelinating disease (C.F.) who had a biopsy in the subcortical white matter of the right hemisphere was noted to have spontaneous clonic movements of the left lower extremity which occurred intermittently for a few days following surgery. His left leg had already been paralyzed as a result of disease. Repeated examination revealed no change in reflexes or sensory impairment. The clonus resolved within a week, and the patient’s neurologic status then remained the same as it was prior to surgery. Another patient (P.F.) with subacute demyelinating disease and diabetes was in a stage of stupor and quadriplegia prior to biopsy. Following the biopsy procedure, her state of consciousness continued to deteriorate as it had prior to surgery. However, there did not appear to be any evidence that the relentless progression of her clinical course had actually been hastened by surgery. One other patient (J.C.) was in a vegetative state from his presenile dementia; following a biopsy procedure he continued to deteriorate and died the following week. Except for these three patients who continued to deteriorate after surgery, the other 19 patients were either unchanged by the surgical procedure or in many cases were improved (particularly in patients with intention tremor or severe “rubral” tremor secondary to multiple sclerosis on whom thalomatomy was performed at the time of biopsy). Success-failure rate of growing brain cells in culture: comparison of tissues acquired at biopsy, early autopsy, and lute autopsy The clinical and pathological features of patients from whom biopsy and autopsy tissue was acquired are shown in tables 1-3. Various numbers of explants were
Pa-
2 yr history of mental de!.erioration
54
M.H
‘p1i s
Dementia, hyperreflexia, Presenile dementia and bilateral Babiqski
Glioma
seizure. Positive brain scan in right temporal area
Prolonged postictal confusion
2 day history of a
U
M.B
Dystonia Musculorum Deformans
29
D.H
Dystonic posturing of extremities and trunk. Convex scoliosis to the right
U
T.S.
16 yr history of involuntary movements, primarily right-sided
Sudden onset of seizures, psychosis. and weakness
16
S.S.
? virus encephalitis Normal brain
Temperature 105OF coma. pyramidal signs
1/1
Gray and white
Gray and white mattw
Temporal lobe
A strocytoma
Normal
1-4?/4
Gray and white matter of right frontal lobe IJ
414
3/4
114
matter of the temporal lobe
015
Gray matter
11/14
5/5
Total explants
survived
Expl an t s that
Gray and white matter of right frontal lobe
U
tissue
Presenile dementia
Presenile dementia
Gray and white matter
Biopsy tissue too scanty for diagnosis
Global dementia, apraxia. aphasia
Moderate mental Huntington’s impairment. Involuntary chorea movements of head, trunk, and extremities. Pneumoencephalogram demonstrated cortical atrophy
4 yr history of progressive mental impairment
64
J.C.
White matter
Area explanted
Demyelinating disease, probable multiple sclerosis
Demyelinating disease, probable multiple sclerosis
Demyelinating disease, probable multiple sclerosis
Ataxia, nystagmus, temporal pallor, paraplegia-in-flexion, hyperreflexia, bilateral Babinski’s horizontal gaze paresis, decreased proprioception and vibratory sense in legs
Pathologic diagnosis
Spastic quadriparesis, legs greater than arms, loss of all sensory modalities in extremities. bilateral optic atrophy
Clinical diagnosis
Neurological signs
10 yr history of mental changes and involuntary movements. Mother and sister had Huntington’s chorea
3% yr history of slow progressive weakness, sphincter symptoms; 2 yrs of progressive bilateral blindness
56
C.F.
H i s tory
Juvenile diabetic with 2 yrs of unremitting incoordination, urinary incontinence. weakness, and seizures
Sex
17
Age
P.F.
tient
Clinical and pathological feutures of patients f r o m whom biopsy tissue w a s acquired
TABLE 1
r
>
4
R
L
2
to
(D
E3
25-30 yr history of alcoholism; 13 yrs of involuntary movements. 9 yrs of progressive mental difficulties. Mother had dementia
“Restless movements” for yrs. No mental symptoms. Maternal grandfather, mother and 2 brothers with Huntington’s disease
5 day history of upper respiratory infection, vomiting. fever. and seizures
Tremors of all 4 extremities of ’? length. Both mother and sister previously diagnosed a s having essential tremor
U
F
M
F
M
F
55
26
56
2
47
45
T.W.
W.H.
M.B.
M.H.
G.M.
L.K.
U
A flu-like syndrome, nausea, vomiting, and photophobia
2 yr history of mental changes. Aunt died in a mental institution. Parental history not known
F
57
A.Z.
2-? yr history of progressive mental deterioration
M
43
GK
? viral encephalitis
Behavioural changes, seizures. and cervical adenopathy
Enrephalopathy. ? herpes simplex encephalitis
Essential (familial) tremor
Statico-action tremor in both h a n d s and feet Voice “tremulous.” No other sign s
U
Suspect herpes simplex encephalitis
Lethargy, nuchal rigidity, otitis, and eventual coma; CSF pleocytosis: 70 cells. 80‘; mononuclear
Involuntary movements: Huntington’s face greater than chorea extremities. No mental changes
Huntington‘s chorea
Possible Huntington’s chorea
Mental state: indifference. psychomotor retardation. No dementia. Constant chewing movements of mouth. No other involuntary movements. Pneumoencephalogram revealed diffuse ventricular dilation with cerebral atrophy
C horeiform movements, dysarthria. dementia. Pneumoencephalogram revealed hydrocephalus ex vacuo
Prrseiiile dc:nentia
Dementia, perseve. :Ition. positive snout reflex. positive glabellar reflex; pn eu moencep h alogr a m revealed cerebral atrophy
U
U
Herpes simplex encephalitis
U
U
5/5
3/4
Gray and white matter of right frontal lobe
U
2/2
3/3
414
Gray and white matter of right frontal lobe
Gray and white matter of right frontal lobe
Gray and white matter of right frontal lobe
Gray and white matter
Gray and white matter of right frontal lobe
U
U
Gray ,>:id white 111;I tter
Normal
CD
(D
~
m
tJ
r 1 C
0 C
I
9 month history of progressive speech problems and righ t-sid ed weakn e s s
55
EM
-
Seizures. mental changes. and ataxia
15
SK
U. U n k n o w n information
Left hemiparesis. N o sensory signs.
Apoplectic onset of‘ left-sided paralysis 2 yrs ago
38
M F
Right-sided weakness and decreasing state of con sciou m e s s
Dementia and ataxia. candle guttering on pn eu moenc ep h alogram
Dementia. mute. left-sided weakness a i d hyperrcflexia
6 month history of depression and withdrawal. associated with left-sided weakncss, difficulty with speech and urinary incontinence
G M )
Same a s above ( s e e
-
Neurologicnl signs
26
HF
Same a s above ( s e e (G M )
History
Ataxia. dementia. and myochonic seizures
4
N Y
Sex
11’2-2 hr history of seizures, mental changes. and impaired balance
47
Age
G M
Patienl
Gray and white 111a tter Left frontal lobe
Gliosis
Low grade glioina
Tuberous sclerosis
Focal left hemispheric destructive lesion, rule out glioma
Gray and white matter of frontal lobe
U
Cerebral itifarctioil right hemisphere
Gray and white matter
Gray and white matter of right frontal lobe
A r e a expl;tntrd
Gray and white matter
Batten’s disease
U
Patholopic di;igliosis
Normal brain
Diffuse enc c p h al o p a t h y
Possible lipoidosis “myoclonic epilepsy”
Essential (familial) tremor
C1iiric;il diagncisia
t5
Total explnnts
survived
th.tt
E XI1 l:lll
c3 0 0
H U M A N BRAIN I N TISSUE C U L T U R E
made from each brain received, and each petri dish or T-25 Falcon flask in which growth of tissue was attempted was listed as a n independent explant. Success was achieved in the attempt to grow tissue from nearly every brain specimen acquired at biopsy or early autopsy regardless of the disease. Table 4 summarizes the total explants acquired from either biopsy, early autopsy (less than six hours after death), or late autopsy (more than six hours after death). Eighty percent of all biopsied tissue (regardless of disease) was successfully grown as a monolayer in culture. In addition, nearly two-thirds of all tissue acquired in less than six hours after death was successfully grown, whereas only one-third of all tissue acquired more than six hours after death was successfully grown in tissue culture. Furthermore, although in some instances biopsy tissue was immediately placed into tissue culture medium as described in MATERIALS AND METHODS, the brain tissue was not explanted until 1620 hours later; yet the success rate in growing this biopsy tissue was still high. In fact, brain tissue was often obtained in another city, placed into tissue culture medium, brought to our laboratories and not explanted until the following morning, nearly one day after it was removed from the patient, yet excellent growth was still obtained.
Explanting us. trypsinization Both explanting and trypsinization techniques were initially performed in this laboratory to establish monolayers in culture. Once small fragments of brain tissue adhered to the petri dish or flask, cell proliferation usually occurred. Cell monolayers were generally not formed until 2-3 weeks later. The total number of monolayers established from explants or from trypsinized tissue was similar. Cells that started to proliferate only 3 4 weeks after seeding of brain tissue generally never grew adequately to form a confluent monolayer, and the procedure was considered unsuccessful if there was no cell growth by one month. Ability to grow tissue f r o m various areas of normal and diseased brain It took longer to produce cell monolayers after explanting gray matter than white matter. Ultimately, however, the same
30 1
uniformity of success was achieved growing monolayers from both gray matter and white matter. Cells from tissue fragments removed from the center of a chronic multiple sclerosis plaque visible to the naked eye rarely grew out, and, even then, cell growth was usually poor. Tissue from grossly normal appearing white matter of multiple sclerosis brain grew a s well as white matter of brain tissue from patients without other neurologic disease. Cells from tissue that was removed from the junction of plaque and white matter areas (periplaque) grew with considerable variability (fig. l ) , and often it was difficult to establish a monolayer from this area. If, however, cells from periplaque areas did grow out, the growth was as abundant a s if the material had been removed from an area appearing normal. Cell monolayers were regularly established from brain tissue of patients with gray matter disease (tables 1-3) such as Huntington’s chorea, motorneuron disease, and the presenile dementias. Cell monolayers were easily established in less than one week from either the choroid plexus or optic nerve. Growth from these areas was always faster than from brain parenchyma. Their different morphologies are described in another study (Rorke et al., ’75).
Effect of various p H , various FCS concentrations, and frequency of m e d i u m changes o n establishment of monolayer cultures Although no detailed studies were done to compare the effect of the pH of the medium on the ability to establish monolayers in culture, cells grew out from both biopsy and autopsy fragments explanted a t pH’s of 7.2, 7.6, or 8.0. No attempts were made to grow cells in medium at a pH lower than 7.0. Of considerable importance was the concentration of FCS in the growth medium. The ability to achieve a monolayer depended directly on whether brain tissue adhered to the glass or plastic of the culture vessel and a 50 % concentration of FCS was found optimal for such adherence. When lesser concentrations of FCS were used, brain fragments were easily dislodged from the bottom of the flask. Leaving either explanted or trypsinized
Optic pallor, nystagmus, left palatal weakness, slurred speech, weakness, atrophy, and decreased proprioception
9 month history of difficulty walking, progressive weakness, a n d diplopia
38
49
R.W.
J.H.
G.K. 44
Muscle weakness a n d atrophy, hyporeflexia, fasiculations
3 yr history of weakness, falling, wasting, a n d weight loss
63
Tremor, quadriplegia, optic atrophy, and nystagmus
U
17 yr history of progressive visual impairment, weakness, paralysis, a n d sphincter symptoms
U
Multiple sclerosis
Normal brain
Suspect multiple sclerosis
Multiple sclerosis
Multiple sclerosis
Multiple sclerosis
Motor neuron disease
Motor neuron disease
Presenile dementia
Presrnile dementia
Global dementia, apraxia, aphasia
V.W.
progressive mental impairment
Post-infectious encephalomy el i t i s
Pathologic diagnosis
Demyelinating disease, ? multiple sclerosis
Clinic a1 diagnosis
Inappropriate laughter, nystagmus, myelopathy with TILT2 level
4 yr history of
Neurological signs
64
parasthesias i n legs and trunk, sphincter and sexual impairment, decreased memory
6 month history of
History
J.C.
Sex
36
Age
E.N.
Patient
5
3-4
1
2
3-4
5
after death until explant
Hrs.
Ponsand subcortical whitq matter
White matter: plaques, periplaque, and “normal” white matter
White matter: plaques, periplaque, a n d “normal” white matter
Cerebrum and spinal cord, both gray and white matter
White and gray matter, left parietal lobe
Periplaques in parietal lobe, pons, a n d spinal cord
Area
explanted
Clinicul and pathologicul f e a t u r e s of p a t i e n t s f r o m w h o m tissue w a s acquired at ecirly a u t o p s y (less t h a n 6 hours ufter d e a t h )
TABLE 2
8/14
18/18
16/21
Total
explants
Explan t s that survived
Acute viral encephalitis
Lethargy, coma, pyramidal signs, and blindness
1 week of personality changes, blindness, and paralysis
F
18
52
52
J.O.
M.G.
E.W.
Multiple sclerosis
Multiple sclerosis
Multiple sclerosis
Multiple sclerosis
Spastic-ataxic gait, hyperreflexia, bilateral Babinski’s, decreased proprioception and vibratory sense in the feet Ophthalmoplegia, bilateral scotomata, ataxia, spasticity. hyperreflexia, Babinski’s signs bilaterally, hypalgesia and proprioceptive loss
6 y r history of progressive weakness in legs and hands with parethesias and pain in all 4 extremities
20 yr history of frequent falling, double vision, weakness and progressive paralysis
U. Unknown information
F
F
SSPE
Acute hemorrhagic leukoencephalitis
Acute necrotizing leukoencephalopathy
Coma, flaccid quadriparesis, increased CSF pressure, 900 RBC’s, CSF protein 389
2daysofheadache and right-sided seizures and weakness
M
15
M.D.
Atrophy, wasting hyperreflexia, and fibrillation on EMG
1-2 yr history of weakness and paralysis
F
50
M.B.
F
A typical motor neuron disease
Motor neuron disease
Motor neuron disease
Motor neuron disease
Wasting. atrophy, fasiculations of tongue and extremities. areflexia
sclerosis, (2) Polio encephalitis of amygdaloid nucleus
(1) Multiple
Multiple sclerosis
Normal
2 yr history of progressive weakness, wasting, dysarthria, and dysphagia
Nystagmus. ptosis, tremor of trunk and extremities, paraparesis, atrophy, hyperreflexia, bilateral B abinski’s decreased vibratory and position sense, decreased pain sensation u p to T8
Suspect multiple sclerosis
44
M.M.
F
13 y r history of progressive weakness, incoordination, sphincter symptoms
Nystagmus, left-sided ataxia, and hyperreflexia
74
H.E.
F
Personality changes
35
i.D.
1-2
2-3
1-2
5
1-2
4
19/19
18/20
Periplaques
016
313
718
0115
12/18
13/26
Plaques, periplaques, and “normal” white matter
Temporal lobe white matter
Left frontal lobe (grossly normal), left occipital lobe (hemorrhagic)
Gray and white matter
Pons
Medulla and cervical cord; frontal lobe
Periplaque
0 0 0
c
1
M
J.J. I 46
Multiple sclerosis
Multiple sclerosis
Right abducens paralysis, vertical nystagmus. cerebellar ataxia, and hyperreflexia Left temporal disc pallor, decreased right corneal reflex, right central facial weakness, left palatal weakness, decreased pain and vibratory sensation in legs
Alcoholism
Presenile dementia. possible variant of JakobCreutzfeldt
Clinic a1 d iagn o s1s
See history
Dementia, bilateral pyramidal signs, extrapyramidal syndrome, and in yoclon u s
Neurological s i g n s
Multiple sclerosis
Multiple sclerosis
None
Presenile dementia
Pathologic diagnosis
M a t e r i a l received from California: c o n t a m i n a t e d w i t h f u n g i . m a t e r i a l treated w i t h antibiotics a n d Amphotericin.
19 yr history of diplopia, paresthesia, and urgency with urgency incontinence
12 month history of dizziness, loss of balance. progressive visual loss, weakness paralysis, and coma
M
20
R.M.
18 month history of dementia, onset of gait disturbance and weakness a few months before death
History
Alcoholic found dead at home
39
R.N.
F
Sex
M
45
Age
P.S.
Patient
14
6-7
8-9
24
plant
ex^
tlrs. after death u n ti 1
plaques
White old andmatter. fresh
Plaques in frontal and parietal lobe and pons
White matter
Gray and white matter
Area
ex planted
116
Oi 8
313
313
Total ex pl an t s
E xplan t 6 that survived
A
0
w
H U M A N BRAIN IN TISSUE C U L T U R E TABLE 4
E s t u b l i s h i n y m o n o l a y e r s of b r a i n t i s s u e i n cultiire: comptrrntiue success from b i o p s y , ecirly a u t o p s y , or l a t e a u t o p s y Total explants
Biopsy Early autopsy 1 Late autopsy 2 1
2
83 190
20
Number of Percent of monolayers monolayers established established
67 119 7
80
63 35
Less than 6 hrs after death 6-24 hrs after death
tissue culture material in the incubator undisturbed for at least seven days even without replacement of medium was critical for the successful outgrowth of cells. Once the tissue adhered to glass or plastic and cell proliferation was noted at the edge of each adhering fragment, medium was replaced. Thereafter medium was changed every 7-10 days depending on the rate of cell growth. Replacement of the medium by half volume of “spent” medium from the old culture and half volume of fresh medium did not have any apparent effect on the eventual outgrowth of the cells.
T h e effect of storage on the recovery of cells Growth of cells from brain tissue which had been frozen for any period of time could not be achieved. It has been possible, however, to recover viable cells from a monolayer culture that had been stored in frozen state. Recovery of these cells is described in a separate study (Wroblewska et al., ’75). DISCUSSION
These studies establish the relative ease with which human brain tissue can be grown in culture. One of the critical features on which success is based is the time which elapses from the death of the patient until the brain tissue is placed in culture medium. The fact that both brain biopsy and autopsy tissue placed in culture medium immediately after removal from the head will still grow if explanted the next day demonstrates that the medium used provides enough nutrient support for eventual cell growth. The four cases in table 3 (acquired at late autopsy) are too few for meaningful statistical analysis: the fact that 100% of the explants from cases P.S.
305
and R.N. survived obscures the fact that repeated attempts at explanting tissue at late autopsy were unsuccessful, and thus, careful records from each late autopsy specimen acquired were intentionally not maintained except for the four patients listed. Cell growth from biopsy fragments from both gray and white matter of patients with chronic neurologic disease involving either gray or white matter was successful. Since in most cases, despite both the underlying chronic neurologic disease and the neurosurgical procedure involved in biopsy, a permanent increase in clinical neurologic deficit of the patient does not appear to develop, this procedure could be used for investigative studies dependent upon the growth of brain cells in culture, particularly when biopsy could be of potential benefit to patients with chronic neurologic disease. A drawback to the biopsy procedure is that a normal area of brain may be sampled. This is particularly important in cases of multiple sclerosis where it may be important to grow cells from the plaque border. For this reason, in multiple sclerosis, autopsied tissue is preferred for extensive virologic studies rather than biopsy tissue which in two cases was found to be derived from histologically normal white matter. Based on our results with both trypsinizing and explanting techniques, we prefer the latter procedure. The comparative ease with which explants are carried out makes it possible to process 15-20 specimens by explanting in the same time consumed in trypsinizing 3-4 brain specimens. Others have reported cell growth in one week by trypsinizing SSPE brain tissue (Chen et al., ’69), but we find that this is the exception, and it usually takes 2-3 weeks before monolayers are established after seeding trypsinized tissue. This could reflect slight differences in the trypsinization procedure. High concentrations of FCS are necessary for cells to begin growing. While a lesser concentration of FCS may be adequate (Ponten, ’73), we have obtained better tissue growth in 50% FCS. This concentration of FCS probably permits better adherence of tissue to glass and plastic, and may represent no more than a modification of the former use of chicken plasma clot explants for in vitro use (Parker, ’61). Embedding tissue fragments in plasma clots is cum-
GILDEN ET AL
306
bersome, however, while explanting them in 50% FCS is simple. Further indications that adherence to glass and plastic plays a n important role in establishing a brain culture may come from our observations that frequent medium changes of explant cultures is not helpful and may sometimes prevent tissue clumps from adhering to the glass or plastic. However, others have replaced the medium for growth of “glial” tumor cells 1-2 days after explanting and 2-3 times weekly thereafter with good success (Ponten and M a c h tyre, ’68). Finally, it must be emphasized that if growth of brain tissue in culture is desired, the material must be explanted or trypsinized fresh. Attempts to grow brain in culture which was previously kept at - 80°C were unsuccessful. ACKNOWLEDGMENTS
The authors are deeply indebted to Drs. Stanley Stellar, Glenn Smith, Irving Cooper, Labe Scheinberg, Lucien Rubinstein, and John Whittaker for their help in supplying our laboratory with brain specimens from a wide spectrum of neurologic disorders. We also acknowledge the expert technical assistance of Ms. Mary Wellish, Ms. Marilyn Chesler, and Mr. Craig Bogen. We also appreciate the constructive comments of Dr. V. J. Cristofalo who reviewed this manuscript.
This research was supported in part by Public Health Service Research grants NS 11036 and NS 09779 from the National Institute of Neurological Diseases and Stroke, RR 05540 from the Division of Research Resources, a grant from the John A. Hartford Foundation, Inc., and a grant from the National Multiple Sclerois Society. LITERATURE CITED Chen, T. T., I . Watanabe, W . Zeman and J. Mealey 1969 Subacute sclerosing panencephalitis: propagation of measles virus from brain biopsy in tissue culture. Science, 163: 1193-1194. Croce, C. M., H. Koprowski and H. Eagle 1972 Effect of environmental pH on the efficiency of cellular hybridization. Proc. Nat. Acad. Sci. U.S.A., 69: 1953-1956. Parker, R. C . 1961 Methods of Tissue Culture. Third ed. Paul B. Hoeber, Inc., New York. Peters, G. 1968 Multiple sclerosis. In: Pathology of the Nervous System. Volume I . J. Minkler, ed. McGraw Hill, New York, pp. 821-843. Ponten, J. 1973 H u m a n glia cells. I n : Tissue Culture Methods and Applications. P. S. Kruse and M. K. Patterson, Jr., e d s . Academic Press, Inc., New York, p. 50. Ponten, J., and E. H. Macintyre 1968 Long term culture of normal and neoplastic human glia. Acta Path. Microbiol. Scandinav., 74: 46-86. Rorke, L. B . , D. H. Gilden, Z . Wroblewska and D. Santoli 1975 Human brain in tissue culture. I V . Morphological characteristics. J . Comp. Neur., I61 : 329-340. Wroblewska, Z . , M. Devlin, D. H. Gilden, D. Santoli, H. Friedman and H. Koprowski 1975 Human brain in tissue culture. 11. Studies of long-term cultures. J. Comp. Neur., 161: 307316.
This paper details the i n vitro techniques used to establish cells ABSTRACT in culture froin the brains of 40 patients, most of whom had chronic neurologic disease. The clinical and pathologic features of these patients are given. The success in establishing cell lines was dependent upon the origin of tissue (biopsy vs. autopsy), the site of removal from the brain, and various environmental and technical manipulations in vitro.
The search for transmissible or viral agents in patients with multiple sclerosis and other chronic neurologic diseases necessitated the acquisition of considerable quantities of brain tissue for growth in vitro. Within the past three years, the Wistar Institute has accumulated brain specimens at biopsy or autopsy of 40 patients with multiple sclerosis, amyotrophic lateral sclerosis, Huntington’s chorea, dystonia musculorum deformans, Batten’s disease, tuberous sclerosis, intention tremor, alcoholism, glioma, acute viral encephalitis (including herpes simplex encephalitis), subacute sclerosing panencephalitis (SSPE), post-infectious encephalomyelitis, acute hemorrhagic leukoencephalitis, and brain tissue from patients with or without systemic disease. This paper details the acquisition and initial handling of these tissues and includes a description of the effect of biopsy on the clinical course of patients with chronic neurologic disease. Also described are the success and failure rates of establishing brain cell monolayers in culture, comparing explanting and trypsinizing techniques in both biopsy- and autopsyacquired tissue, the ability of tissue from various areas of the brain (gray or white matter, “normal” or diseased areas, plaques in multiple sclerosis brain, and gray matter from patients with primary gray matter disease) to grow in culture, the establishment of initial cultures from non-parenchymal J . COMP. NEUR.,1 6 1 : 295-306.
areas of brain (choroid plexus and optic nerve), observations on the effect of pH, and various fetal calf serum (FCS) concentrations on the formation of monolayers, the effect of changes in nutrient medium on initial cultures, and the effect of specimen storage on the ability to grow cells out of brain tissue. MATERIALS AND METHODS
Acquisition of brain tissue. All biopsy material was removed by a neurosurgeon in the operating room under sterile conditions and immediately placed in tissue culture medium. Autopsy was performed as soon a s possible after death. Tissue culture media. The medium consisted of Eagle’s minimal essential medium (MEM) with phenol red and a 2 X concentration of vitamins. In general, the medium was adjusted at pH 7.2 by addition of 5.6% sodium bicarbonate. When it was desired to keep the medium at pH 8.0 or pH 7.6, MEM buffered with organic buffers and sodium bicarbonate was used (Croce et al., ’72). FCS which had been inactivated for 30 minutes at 56°C (Flow Laboratories, Rockville, Maryland) was added to produce a final concentration of l o % , and L-glutamine (Flow Laboratories, Rockville, Maryland) was added to a final concentration of 0.5%. Penicillin was added to produce a final concentration of 100 unitslml and Streptomycin was added to produce a final concentration of 0.05 mg/ml. 295
296
G I L D E N ET A L
Autopsy procedure. Following removal of the brain from the skull under clean but not sterile conditions, and with the dura intact, the brain was placed with the base down on a clean towel. At this point the dura was carefully removed and the convexities were wiped with 70% alcohol. A long brain cutting knife was then dipped into 100% alcohol and flamed until dry. After 15 to 20 seconds had been allowed for the knife to cool, a coronal section was made through the frontal region of the brain. Appropriate sections were then picked out of the cortex (gray matter just beneath the surface) and separate pieces of subcortical white matter were sampled. This material was kept sterile and immediately placed in tissue culture media. Then a more posterior area of the convexities was wiped with 70% alcohol, the brain cutting knife again sterilized in 100% alcohol, and the entire procedure repeated. Multiple coronal sections through the brain and brain stem were made in this fashion.
Multiple scleroszs brain. Old plaque areas of multiple sclerosis brain were readily identifiable by their brownish color, sharp borders, and varying degrees of tissue retraction (Peters, ’68). Acute plaques were more difficult to visualize because of their lighter salmon color, irregular borders, and absence of retraction (Peters, ’68). Small 1-2 mm’ sections were removed from the center of the plaque, the junction of plaque and white matter (periplaque), and grossly “normal” white matter 1-2 cm from the plaque. White matter material was sampled from both fresh and old plaques (fig. 1) and also gray matter. Each specimen was processed separately. Explants. Although the sizes of the brain specimens placed in tissue culture medium varied, no more than 1 m m 3of tissue was usually removed. All explanting was done in a laminar flow hood (Biogard, Baker Co., Inc., Sanford, Maine). The brain specimen was cut into as small pieces as could be cut with sterile knives and the
Fig. 1 Grossly visible plaques in the cerebellar white matter of a patient with multiple sclerosis. Note chronic plaque (above) with dark rusty appearance and sharp borders in contrast to fresh plaque (below) with lighter salmon color and hazy borders. Plaque-white matter j u n c tion noted by short arrow and periplaque white matter noted by long arrows.
297
H U M A N BRAIN I N TISSUE C U L T U R E
pieces were washed once or twice in tissue culture medium, and seeded sparsely into a small glass petri dish and at least 2 T-25 plastic Falcon flasks in MEM with 50% FCS. No less than three explants were made from each specimen removed. The T-25 flasks were placed in a 37°C incubator with 4%-5% C 0 2 . Caps were kept loose all the time until a monolayer was achieved. Medium was not changed until one week after the initial explant was placed in the culture flask or petri dish. Further medium changes were done at approximately 7-10 day intervals thereafter. Trypsinization. Brain tissue to be trypsinized was rinsed twice in tissue culture medium (MEM plus 10% FCS) and placed in a 35 ml trypsinization flask (Bellco Glass Inc., Vineland, New Jersey). Trypsin was then diluted to a final concentration of 0.25% in phosphate buffered saline and FCS was added to make a final concentration of 3% (trypsin was purchased from Flow Laboratories, Rockville, Maryland, and was kept at a 2.5% concentration in modified Hank’s balanced salt solution without calcium and magnesium salts). This brain tissue was stirred for 15 minutes at room temperature. The supernatant was then removed and kept at 4 “C. An additional 10 ml of 0.25% trypsin was added to the brain tissue which remained and stirred again for 15 minutes at room temperature. The second supernatant was removed and pooled with the first. Both were kept at 4°C and the entire procedure was repeated a third time. All the suspended cellular material was spun at 800-1000 rpm for 5 minutes. The supernatant was removed and the pellet was resuspended in MEM plus 50% FCS. This entire material was then filtered through a nylon mesh and distributed to tissue culture flasks. All explanted and trypsinized brain tissue was kept in MEM with 50% FCS for no less than two weeks (at least one medium change). Thereafter, 50% FCS was replaced by 20% FCS until a monolayer was established. Lesser concentrations of FCS were not used in this laboratory until after the first cell transfer. All old medium was removed during each medium change, or half the volume of “spent” medium was removed and replaced with fresh medium. All explanting and trypsinizing procedures were performed in a laminar flow hood.
RESULTS
T h e effect of biopsy on the clinical course of patients In 19 out of 22 patients on whom biopsies were performed, there was no adverse effect on the clinical course of the patient. Most patients already had considerable neurologic deficit as a result of their disease (tremor, paralysis, dementia, and sometimes coma), and a n increase in neurologic signs was not observed after biopsy. One patient with demyelinating disease (C.F.) who had a biopsy in the subcortical white matter of the right hemisphere was noted to have spontaneous clonic movements of the left lower extremity which occurred intermittently for a few days following surgery. His left leg had already been paralyzed as a result of disease. Repeated examination revealed no change in reflexes or sensory impairment. The clonus resolved within a week, and the patient’s neurologic status then remained the same as it was prior to surgery. Another patient (P.F.) with subacute demyelinating disease and diabetes was in a stage of stupor and quadriplegia prior to biopsy. Following the biopsy procedure, her state of consciousness continued to deteriorate as it had prior to surgery. However, there did not appear to be any evidence that the relentless progression of her clinical course had actually been hastened by surgery. One other patient (J.C.) was in a vegetative state from his presenile dementia; following a biopsy procedure he continued to deteriorate and died the following week. Except for these three patients who continued to deteriorate after surgery, the other 19 patients were either unchanged by the surgical procedure or in many cases were improved (particularly in patients with intention tremor or severe “rubral” tremor secondary to multiple sclerosis on whom thalomatomy was performed at the time of biopsy). Success-failure rate of growing brain cells in culture: comparison of tissues acquired at biopsy, early autopsy, and lute autopsy The clinical and pathological features of patients from whom biopsy and autopsy tissue was acquired are shown in tables 1-3. Various numbers of explants were
Pa-
2 yr history of mental de!.erioration
54
M.H
‘p1i s
Dementia, hyperreflexia, Presenile dementia and bilateral Babiqski
Glioma
seizure. Positive brain scan in right temporal area
Prolonged postictal confusion
2 day history of a
U
M.B
Dystonia Musculorum Deformans
29
D.H
Dystonic posturing of extremities and trunk. Convex scoliosis to the right
U
T.S.
16 yr history of involuntary movements, primarily right-sided
Sudden onset of seizures, psychosis. and weakness
16
S.S.
? virus encephalitis Normal brain
Temperature 105OF coma. pyramidal signs
1/1
Gray and white
Gray and white mattw
Temporal lobe
A strocytoma
Normal
1-4?/4
Gray and white matter of right frontal lobe IJ
414
3/4
114
matter of the temporal lobe
015
Gray matter
11/14
5/5
Total explants
survived
Expl an t s that
Gray and white matter of right frontal lobe
U
tissue
Presenile dementia
Presenile dementia
Gray and white matter
Biopsy tissue too scanty for diagnosis
Global dementia, apraxia. aphasia
Moderate mental Huntington’s impairment. Involuntary chorea movements of head, trunk, and extremities. Pneumoencephalogram demonstrated cortical atrophy
4 yr history of progressive mental impairment
64
J.C.
White matter
Area explanted
Demyelinating disease, probable multiple sclerosis
Demyelinating disease, probable multiple sclerosis
Demyelinating disease, probable multiple sclerosis
Ataxia, nystagmus, temporal pallor, paraplegia-in-flexion, hyperreflexia, bilateral Babinski’s horizontal gaze paresis, decreased proprioception and vibratory sense in legs
Pathologic diagnosis
Spastic quadriparesis, legs greater than arms, loss of all sensory modalities in extremities. bilateral optic atrophy
Clinical diagnosis
Neurological signs
10 yr history of mental changes and involuntary movements. Mother and sister had Huntington’s chorea
3% yr history of slow progressive weakness, sphincter symptoms; 2 yrs of progressive bilateral blindness
56
C.F.
H i s tory
Juvenile diabetic with 2 yrs of unremitting incoordination, urinary incontinence. weakness, and seizures
Sex
17
Age
P.F.
tient
Clinical and pathological feutures of patients f r o m whom biopsy tissue w a s acquired
TABLE 1
r
>
4
R
L
2
to
(D
E3
25-30 yr history of alcoholism; 13 yrs of involuntary movements. 9 yrs of progressive mental difficulties. Mother had dementia
“Restless movements” for yrs. No mental symptoms. Maternal grandfather, mother and 2 brothers with Huntington’s disease
5 day history of upper respiratory infection, vomiting. fever. and seizures
Tremors of all 4 extremities of ’? length. Both mother and sister previously diagnosed a s having essential tremor
U
F
M
F
M
F
55
26
56
2
47
45
T.W.
W.H.
M.B.
M.H.
G.M.
L.K.
U
A flu-like syndrome, nausea, vomiting, and photophobia
2 yr history of mental changes. Aunt died in a mental institution. Parental history not known
F
57
A.Z.
2-? yr history of progressive mental deterioration
M
43
GK
? viral encephalitis
Behavioural changes, seizures. and cervical adenopathy
Enrephalopathy. ? herpes simplex encephalitis
Essential (familial) tremor
Statico-action tremor in both h a n d s and feet Voice “tremulous.” No other sign s
U
Suspect herpes simplex encephalitis
Lethargy, nuchal rigidity, otitis, and eventual coma; CSF pleocytosis: 70 cells. 80‘; mononuclear
Involuntary movements: Huntington’s face greater than chorea extremities. No mental changes
Huntington‘s chorea
Possible Huntington’s chorea
Mental state: indifference. psychomotor retardation. No dementia. Constant chewing movements of mouth. No other involuntary movements. Pneumoencephalogram revealed diffuse ventricular dilation with cerebral atrophy
C horeiform movements, dysarthria. dementia. Pneumoencephalogram revealed hydrocephalus ex vacuo
Prrseiiile dc:nentia
Dementia, perseve. :Ition. positive snout reflex. positive glabellar reflex; pn eu moencep h alogr a m revealed cerebral atrophy
U
U
Herpes simplex encephalitis
U
U
5/5
3/4
Gray and white matter of right frontal lobe
U
2/2
3/3
414
Gray and white matter of right frontal lobe
Gray and white matter of right frontal lobe
Gray and white matter of right frontal lobe
Gray and white matter
Gray and white matter of right frontal lobe
U
U
Gray ,>:id white 111;I tter
Normal
CD
(D
~
m
tJ
r 1 C
0 C
I
9 month history of progressive speech problems and righ t-sid ed weakn e s s
55
EM
-
Seizures. mental changes. and ataxia
15
SK
U. U n k n o w n information
Left hemiparesis. N o sensory signs.
Apoplectic onset of‘ left-sided paralysis 2 yrs ago
38
M F
Right-sided weakness and decreasing state of con sciou m e s s
Dementia and ataxia. candle guttering on pn eu moenc ep h alogram
Dementia. mute. left-sided weakness a i d hyperrcflexia
6 month history of depression and withdrawal. associated with left-sided weakncss, difficulty with speech and urinary incontinence
G M )
Same a s above ( s e e
-
Neurologicnl signs
26
HF
Same a s above ( s e e (G M )
History
Ataxia. dementia. and myochonic seizures
4
N Y
Sex
11’2-2 hr history of seizures, mental changes. and impaired balance
47
Age
G M
Patienl
Gray and white 111a tter Left frontal lobe
Gliosis
Low grade glioina
Tuberous sclerosis
Focal left hemispheric destructive lesion, rule out glioma
Gray and white matter of frontal lobe
U
Cerebral itifarctioil right hemisphere
Gray and white matter
Gray and white matter of right frontal lobe
A r e a expl;tntrd
Gray and white matter
Batten’s disease
U
Patholopic di;igliosis
Normal brain
Diffuse enc c p h al o p a t h y
Possible lipoidosis “myoclonic epilepsy”
Essential (familial) tremor
C1iiric;il diagncisia
t5
Total explnnts
survived
th.tt
E XI1 l:lll
c3 0 0
H U M A N BRAIN I N TISSUE C U L T U R E
made from each brain received, and each petri dish or T-25 Falcon flask in which growth of tissue was attempted was listed as a n independent explant. Success was achieved in the attempt to grow tissue from nearly every brain specimen acquired at biopsy or early autopsy regardless of the disease. Table 4 summarizes the total explants acquired from either biopsy, early autopsy (less than six hours after death), or late autopsy (more than six hours after death). Eighty percent of all biopsied tissue (regardless of disease) was successfully grown as a monolayer in culture. In addition, nearly two-thirds of all tissue acquired in less than six hours after death was successfully grown, whereas only one-third of all tissue acquired more than six hours after death was successfully grown in tissue culture. Furthermore, although in some instances biopsy tissue was immediately placed into tissue culture medium as described in MATERIALS AND METHODS, the brain tissue was not explanted until 1620 hours later; yet the success rate in growing this biopsy tissue was still high. In fact, brain tissue was often obtained in another city, placed into tissue culture medium, brought to our laboratories and not explanted until the following morning, nearly one day after it was removed from the patient, yet excellent growth was still obtained.
Explanting us. trypsinization Both explanting and trypsinization techniques were initially performed in this laboratory to establish monolayers in culture. Once small fragments of brain tissue adhered to the petri dish or flask, cell proliferation usually occurred. Cell monolayers were generally not formed until 2-3 weeks later. The total number of monolayers established from explants or from trypsinized tissue was similar. Cells that started to proliferate only 3 4 weeks after seeding of brain tissue generally never grew adequately to form a confluent monolayer, and the procedure was considered unsuccessful if there was no cell growth by one month. Ability to grow tissue f r o m various areas of normal and diseased brain It took longer to produce cell monolayers after explanting gray matter than white matter. Ultimately, however, the same
30 1
uniformity of success was achieved growing monolayers from both gray matter and white matter. Cells from tissue fragments removed from the center of a chronic multiple sclerosis plaque visible to the naked eye rarely grew out, and, even then, cell growth was usually poor. Tissue from grossly normal appearing white matter of multiple sclerosis brain grew a s well as white matter of brain tissue from patients without other neurologic disease. Cells from tissue that was removed from the junction of plaque and white matter areas (periplaque) grew with considerable variability (fig. l ) , and often it was difficult to establish a monolayer from this area. If, however, cells from periplaque areas did grow out, the growth was as abundant a s if the material had been removed from an area appearing normal. Cell monolayers were regularly established from brain tissue of patients with gray matter disease (tables 1-3) such as Huntington’s chorea, motorneuron disease, and the presenile dementias. Cell monolayers were easily established in less than one week from either the choroid plexus or optic nerve. Growth from these areas was always faster than from brain parenchyma. Their different morphologies are described in another study (Rorke et al., ’75).
Effect of various p H , various FCS concentrations, and frequency of m e d i u m changes o n establishment of monolayer cultures Although no detailed studies were done to compare the effect of the pH of the medium on the ability to establish monolayers in culture, cells grew out from both biopsy and autopsy fragments explanted a t pH’s of 7.2, 7.6, or 8.0. No attempts were made to grow cells in medium at a pH lower than 7.0. Of considerable importance was the concentration of FCS in the growth medium. The ability to achieve a monolayer depended directly on whether brain tissue adhered to the glass or plastic of the culture vessel and a 50 % concentration of FCS was found optimal for such adherence. When lesser concentrations of FCS were used, brain fragments were easily dislodged from the bottom of the flask. Leaving either explanted or trypsinized
Optic pallor, nystagmus, left palatal weakness, slurred speech, weakness, atrophy, and decreased proprioception
9 month history of difficulty walking, progressive weakness, a n d diplopia
38
49
R.W.
J.H.
G.K. 44
Muscle weakness a n d atrophy, hyporeflexia, fasiculations
3 yr history of weakness, falling, wasting, a n d weight loss
63
Tremor, quadriplegia, optic atrophy, and nystagmus
U
17 yr history of progressive visual impairment, weakness, paralysis, a n d sphincter symptoms
U
Multiple sclerosis
Normal brain
Suspect multiple sclerosis
Multiple sclerosis
Multiple sclerosis
Multiple sclerosis
Motor neuron disease
Motor neuron disease
Presenile dementia
Presrnile dementia
Global dementia, apraxia, aphasia
V.W.
progressive mental impairment
Post-infectious encephalomy el i t i s
Pathologic diagnosis
Demyelinating disease, ? multiple sclerosis
Clinic a1 diagnosis
Inappropriate laughter, nystagmus, myelopathy with TILT2 level
4 yr history of
Neurological signs
64
parasthesias i n legs and trunk, sphincter and sexual impairment, decreased memory
6 month history of
History
J.C.
Sex
36
Age
E.N.
Patient
5
3-4
1
2
3-4
5
after death until explant
Hrs.
Ponsand subcortical whitq matter
White matter: plaques, periplaque, and “normal” white matter
White matter: plaques, periplaque, a n d “normal” white matter
Cerebrum and spinal cord, both gray and white matter
White and gray matter, left parietal lobe
Periplaques in parietal lobe, pons, a n d spinal cord
Area
explanted
Clinicul and pathologicul f e a t u r e s of p a t i e n t s f r o m w h o m tissue w a s acquired at ecirly a u t o p s y (less t h a n 6 hours ufter d e a t h )
TABLE 2
8/14
18/18
16/21
Total
explants
Explan t s that survived
Acute viral encephalitis
Lethargy, coma, pyramidal signs, and blindness
1 week of personality changes, blindness, and paralysis
F
18
52
52
J.O.
M.G.
E.W.
Multiple sclerosis
Multiple sclerosis
Multiple sclerosis
Multiple sclerosis
Spastic-ataxic gait, hyperreflexia, bilateral Babinski’s, decreased proprioception and vibratory sense in the feet Ophthalmoplegia, bilateral scotomata, ataxia, spasticity. hyperreflexia, Babinski’s signs bilaterally, hypalgesia and proprioceptive loss
6 y r history of progressive weakness in legs and hands with parethesias and pain in all 4 extremities
20 yr history of frequent falling, double vision, weakness and progressive paralysis
U. Unknown information
F
F
SSPE
Acute hemorrhagic leukoencephalitis
Acute necrotizing leukoencephalopathy
Coma, flaccid quadriparesis, increased CSF pressure, 900 RBC’s, CSF protein 389
2daysofheadache and right-sided seizures and weakness
M
15
M.D.
Atrophy, wasting hyperreflexia, and fibrillation on EMG
1-2 yr history of weakness and paralysis
F
50
M.B.
F
A typical motor neuron disease
Motor neuron disease
Motor neuron disease
Motor neuron disease
Wasting. atrophy, fasiculations of tongue and extremities. areflexia
sclerosis, (2) Polio encephalitis of amygdaloid nucleus
(1) Multiple
Multiple sclerosis
Normal
2 yr history of progressive weakness, wasting, dysarthria, and dysphagia
Nystagmus. ptosis, tremor of trunk and extremities, paraparesis, atrophy, hyperreflexia, bilateral B abinski’s decreased vibratory and position sense, decreased pain sensation u p to T8
Suspect multiple sclerosis
44
M.M.
F
13 y r history of progressive weakness, incoordination, sphincter symptoms
Nystagmus, left-sided ataxia, and hyperreflexia
74
H.E.
F
Personality changes
35
i.D.
1-2
2-3
1-2
5
1-2
4
19/19
18/20
Periplaques
016
313
718
0115
12/18
13/26
Plaques, periplaques, and “normal” white matter
Temporal lobe white matter
Left frontal lobe (grossly normal), left occipital lobe (hemorrhagic)
Gray and white matter
Pons
Medulla and cervical cord; frontal lobe
Periplaque
0 0 0
c
1
M
J.J. I 46
Multiple sclerosis
Multiple sclerosis
Right abducens paralysis, vertical nystagmus. cerebellar ataxia, and hyperreflexia Left temporal disc pallor, decreased right corneal reflex, right central facial weakness, left palatal weakness, decreased pain and vibratory sensation in legs
Alcoholism
Presenile dementia. possible variant of JakobCreutzfeldt
Clinic a1 d iagn o s1s
See history
Dementia, bilateral pyramidal signs, extrapyramidal syndrome, and in yoclon u s
Neurological s i g n s
Multiple sclerosis
Multiple sclerosis
None
Presenile dementia
Pathologic diagnosis
M a t e r i a l received from California: c o n t a m i n a t e d w i t h f u n g i . m a t e r i a l treated w i t h antibiotics a n d Amphotericin.
19 yr history of diplopia, paresthesia, and urgency with urgency incontinence
12 month history of dizziness, loss of balance. progressive visual loss, weakness paralysis, and coma
M
20
R.M.
18 month history of dementia, onset of gait disturbance and weakness a few months before death
History
Alcoholic found dead at home
39
R.N.
F
Sex
M
45
Age
P.S.
Patient
14
6-7
8-9
24
plant
ex^
tlrs. after death u n ti 1
plaques
White old andmatter. fresh
Plaques in frontal and parietal lobe and pons
White matter
Gray and white matter
Area
ex planted
116
Oi 8
313
313
Total ex pl an t s
E xplan t 6 that survived
A
0
w
H U M A N BRAIN IN TISSUE C U L T U R E TABLE 4
E s t u b l i s h i n y m o n o l a y e r s of b r a i n t i s s u e i n cultiire: comptrrntiue success from b i o p s y , ecirly a u t o p s y , or l a t e a u t o p s y Total explants
Biopsy Early autopsy 1 Late autopsy 2 1
2
83 190
20
Number of Percent of monolayers monolayers established established
67 119 7
80
63 35
Less than 6 hrs after death 6-24 hrs after death
tissue culture material in the incubator undisturbed for at least seven days even without replacement of medium was critical for the successful outgrowth of cells. Once the tissue adhered to glass or plastic and cell proliferation was noted at the edge of each adhering fragment, medium was replaced. Thereafter medium was changed every 7-10 days depending on the rate of cell growth. Replacement of the medium by half volume of “spent” medium from the old culture and half volume of fresh medium did not have any apparent effect on the eventual outgrowth of the cells.
T h e effect of storage on the recovery of cells Growth of cells from brain tissue which had been frozen for any period of time could not be achieved. It has been possible, however, to recover viable cells from a monolayer culture that had been stored in frozen state. Recovery of these cells is described in a separate study (Wroblewska et al., ’75). DISCUSSION
These studies establish the relative ease with which human brain tissue can be grown in culture. One of the critical features on which success is based is the time which elapses from the death of the patient until the brain tissue is placed in culture medium. The fact that both brain biopsy and autopsy tissue placed in culture medium immediately after removal from the head will still grow if explanted the next day demonstrates that the medium used provides enough nutrient support for eventual cell growth. The four cases in table 3 (acquired at late autopsy) are too few for meaningful statistical analysis: the fact that 100% of the explants from cases P.S.
305
and R.N. survived obscures the fact that repeated attempts at explanting tissue at late autopsy were unsuccessful, and thus, careful records from each late autopsy specimen acquired were intentionally not maintained except for the four patients listed. Cell growth from biopsy fragments from both gray and white matter of patients with chronic neurologic disease involving either gray or white matter was successful. Since in most cases, despite both the underlying chronic neurologic disease and the neurosurgical procedure involved in biopsy, a permanent increase in clinical neurologic deficit of the patient does not appear to develop, this procedure could be used for investigative studies dependent upon the growth of brain cells in culture, particularly when biopsy could be of potential benefit to patients with chronic neurologic disease. A drawback to the biopsy procedure is that a normal area of brain may be sampled. This is particularly important in cases of multiple sclerosis where it may be important to grow cells from the plaque border. For this reason, in multiple sclerosis, autopsied tissue is preferred for extensive virologic studies rather than biopsy tissue which in two cases was found to be derived from histologically normal white matter. Based on our results with both trypsinizing and explanting techniques, we prefer the latter procedure. The comparative ease with which explants are carried out makes it possible to process 15-20 specimens by explanting in the same time consumed in trypsinizing 3-4 brain specimens. Others have reported cell growth in one week by trypsinizing SSPE brain tissue (Chen et al., ’69), but we find that this is the exception, and it usually takes 2-3 weeks before monolayers are established after seeding trypsinized tissue. This could reflect slight differences in the trypsinization procedure. High concentrations of FCS are necessary for cells to begin growing. While a lesser concentration of FCS may be adequate (Ponten, ’73), we have obtained better tissue growth in 50% FCS. This concentration of FCS probably permits better adherence of tissue to glass and plastic, and may represent no more than a modification of the former use of chicken plasma clot explants for in vitro use (Parker, ’61). Embedding tissue fragments in plasma clots is cum-
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bersome, however, while explanting them in 50% FCS is simple. Further indications that adherence to glass and plastic plays a n important role in establishing a brain culture may come from our observations that frequent medium changes of explant cultures is not helpful and may sometimes prevent tissue clumps from adhering to the glass or plastic. However, others have replaced the medium for growth of “glial” tumor cells 1-2 days after explanting and 2-3 times weekly thereafter with good success (Ponten and M a c h tyre, ’68). Finally, it must be emphasized that if growth of brain tissue in culture is desired, the material must be explanted or trypsinized fresh. Attempts to grow brain in culture which was previously kept at - 80°C were unsuccessful. ACKNOWLEDGMENTS
The authors are deeply indebted to Drs. Stanley Stellar, Glenn Smith, Irving Cooper, Labe Scheinberg, Lucien Rubinstein, and John Whittaker for their help in supplying our laboratory with brain specimens from a wide spectrum of neurologic disorders. We also acknowledge the expert technical assistance of Ms. Mary Wellish, Ms. Marilyn Chesler, and Mr. Craig Bogen. We also appreciate the constructive comments of Dr. V. J. Cristofalo who reviewed this manuscript.
This research was supported in part by Public Health Service Research grants NS 11036 and NS 09779 from the National Institute of Neurological Diseases and Stroke, RR 05540 from the Division of Research Resources, a grant from the John A. Hartford Foundation, Inc., and a grant from the National Multiple Sclerois Society. LITERATURE CITED Chen, T. T., I . Watanabe, W . Zeman and J. Mealey 1969 Subacute sclerosing panencephalitis: propagation of measles virus from brain biopsy in tissue culture. Science, 163: 1193-1194. Croce, C. M., H. Koprowski and H. Eagle 1972 Effect of environmental pH on the efficiency of cellular hybridization. Proc. Nat. Acad. Sci. U.S.A., 69: 1953-1956. Parker, R. C . 1961 Methods of Tissue Culture. Third ed. Paul B. Hoeber, Inc., New York. Peters, G. 1968 Multiple sclerosis. In: Pathology of the Nervous System. Volume I . J. Minkler, ed. McGraw Hill, New York, pp. 821-843. Ponten, J. 1973 H u m a n glia cells. I n : Tissue Culture Methods and Applications. P. S. Kruse and M. K. Patterson, Jr., e d s . Academic Press, Inc., New York, p. 50. Ponten, J., and E. H. Macintyre 1968 Long term culture of normal and neoplastic human glia. Acta Path. Microbiol. Scandinav., 74: 46-86. Rorke, L. B . , D. H. Gilden, Z . Wroblewska and D. Santoli 1975 Human brain in tissue culture. I V . Morphological characteristics. J . Comp. Neur., I61 : 329-340. Wroblewska, Z . , M. Devlin, D. H. Gilden, D. Santoli, H. Friedman and H. Koprowski 1975 Human brain in tissue culture. 11. Studies of long-term cultures. J. Comp. Neur., 161: 307316.
